Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI. while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

The role of type 1 and curli fimbriae of Shiga toxin-producing Escherichia coli in adherence to abiotic surfaces

Biofilm formation on abiotic surfaces may provide a source of microbial contamination and may also enhance microbial environmental survival. The role of fimbrial expression by Shiga toxin-producing Escherichia coli (STEC) in biofilm formation is poorly understood. This study aimed to investigate the role of STEC type 1 and curli fimbriae in adhesion to and biofilm formation on abiotic surfaces. None of 13 O157:H7 isolates expressed either fimbrial type whereas 11 of 13 and 5 of 13 non-O157 STEC elaborated type 1 fimbriae and curli fimbriae, respectively. Mutants made by allelic exchange of a diarrhoeal non-O157 STEC isolate, O128:H2 (E41509), unable to elaborate type 1 and curli fimbriae were made for adherence and biofilm assays. Elaboration of type 1 fimbriae was necessary for the adhesion to abiotic surfaces whereas curliation was associated with both adherence and subsequent biofilm formation. STEC O157:H7 adhered to thermanox and glass but poorly to polystyrene. Additionally, STEC O157:H7 failed to form biofilms. These data indicate that certain STEC isolates are able to form biofilms and that the elaboration of curli fimbriae may enhance biofilm formation leading to possible long-term survival and a potential source of human infection.

Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli 086:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli 086:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type I and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.

Characterisation of attaching-effacing Escherichia coli isolated from animals at slaughter in England and Wales

Escherichia coli isolates were recovered from faecal samples taken from cattle, sheep and pigs at slaughter in England and Wales. Isolates (n = 1227) selected at random from this collection were each hybridised in colony dot-blot experiments with an eae gene probe that presumptively identified attaching-effacing E. coli (AEEC). Of the 99 (8.1%) eae positive isolates 72 were of ovine origin, 24 were of bovine origin and three of porcine origin. None were typed as O157:H7 whereas 78 were assigned to 23 serogroups and 21 were untypable. The most frequently isolated eae positive serogroups were O156 (10), O26 (8), O103 (8), O108 (7) O56 (6) and O168 (6) of which serogroups O103 and O156 only were recovered from all three animal species. In tissue culture adherence assays, 36 representatives of eae positive isolates of all serogroups and host of origin tested induced intimate attachment with varying degrees of actin accumulation and pedestal formation in the HEp-2 cells. The identity of the eae type for these 36 was determined by specific PCR and the most prevalent intimin types were caebeta (15), eaegamma (12) and eaeepsilon (4). Isolates were examined by PCR for the presence of other virulence determinants and five possessed stx1 but none possessed stx2. One O115 eaeepsilon isolate possessed cnf1 and 2, hlyA, etpD and katP genes which is a novel combination of virulence determinants.

Role for flagella but not intimin in the persistent infection of the gastrointestinal tissues of specific-pathogen-free chicks by Shiga toxin-negative Escherichia coli O157:H7

Shiga toxin (Stx)-positive Escherichia coli O157:117 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:117 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in E. coli O157:117 isolate NCTC12900. These mutants were used to inoculate (10(5) CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intiminaflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.

The tenant as a customer: can good service improve real estate performance?

Customer Relationship Management (CRM) theory suggests that good customer service results in satisfied customers, who in turn are more likely to remain loyal and recommend the service provider to others. Proponents of good customer service for tenants claim that landlords should see a return on any investment in their customer service, in the form of enhanced real estate performance. This paper begins by reviewing research on customer service returns in other industries. Through consideration of the characteristics of real estate markets, the paper explains how factors such as (inter alia) lease terms, property market cycles, and property type, might determine the relationship between customer service and real estate performance. The paper concludes that further research is needed to isolate specific aspects of customer service that are most appreciated by customers. It suggests that the financial returns which accrue to landlords adopting a customer-focused approach might indeed be quantified, and suggests an appropriate method for such future research.

Summertime stratospheric processes at northern mid-latitudes: comparisons between MANTRA balloon measurements and the Canadian Middle Atmosphere Model

In this paper we report on a study conducted using the Middle Atmospheric Nitrogen TRend Assessment (MANTRA) balloon measurements of stratospheric constituents and temperature and the Canadian Middle Atmosphere Model (CMAM). Three different kinds of data are used to assess the inter-consistency of the combined dataset: single profiles of long-lived species from MANTRA 1998, sparse climatologies from the ozonesonde measurements during the four MANTRA campaigns and from HALOE satellite measurements, and the CMAM climatology. In doing so, we evaluate the ability of the model to reproduce the measured fields and to thereby test our ability to describe mid-latitude summertime stratospheric processes. The MANTRA campaigns were conducted at Vanscoy, Saskatchewan, Canada (52◦ N, 107◦ W)in late August and early September of 1998, 2000, 2002 and 2004. During late summer at mid-latitudes, the stratosphere is close to photochemical control, providing an ideal scenario for the study reported here. From this analysis we find that: (1) reducing the value for the vertical diffusion coefficient in CMAM to a more physically reasonable value results in the model better reproducing the measured profiles of long-lived species; (2) the existence of compact correlations among the constituents, as expected from independent measurements in the literature and from models, confirms the self-consistency of the MANTRA measurements; and (3) the 1998 measurements show structures in the chemical species profiles that can be associated with transport, adding to the growing evidence that the summertime stratosphere can be much more disturbed than anticipated. The mechanisms responsible for such disturbances need to be understood in order to assess the representativeness of the measurements and to isolate longterm trends.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

Large-scale atmospheric dynamics of the wet winter 2009–2010 and its impact on hydrology in Portugal

The anomalously wet winter of 2010 had a very important impact on the Portuguese hydrological system. Owing to the detrimental effects of reduced precipitation in Portugal on the environmental and socio-economic systems, the 2010 winter was predominantly beneficial by reversing the accumulated precipitation deficits during the previous hydrological years. The recorded anomalously high precipitation amounts have contributed to an overall increase in river runoffs and dam recharges in the 4 major river basins. In synoptic terms, the winter 2010 was characterised by an anomalously strong westerly flow component over the North Atlantic that triggered high precipitation amounts. A dynamically coherent enhancement in the frequencies of mid-latitude cyclones close to Portugal, also accompanied by significant increases in the occurrence of cyclonic, south and south-westerly circulation weather types, are noteworthy. Furthermore, the prevalence of the strong negative phase of the North Atlantic Oscillation (NAO) also emphasises the main dynamical features of the 2010 winter. A comparison of the hydrological and atmospheric conditions between the 2010 winter and the previous 2 anomalously wet winters (1996 and 2001) was also carried out to isolate not only their similarities, but also their contrasting conditions, highlighting the limitations of estimating winter precipitation amounts in Portugal using solely the NAO phase as a predictor.

The experimetrics of public goods: inferring motivations from contributions

In public goods experiments, stochastic choice, censoring and motivational heterogeneity give scope for disagreement over the extent of unselfishness, and whether it is reciprocal or altruistic. We show that these problems can be addressed econometrically, by estimating a finite mixture model to isolate types, incorporating double censoring and a tremble term. Most subjects act selfishly, but a substantial proportion are reciprocal with altruism playing only a marginal role. Isolating reciprocators enables a test of Sugden’s model of voluntary contributions. We estimate that reciprocators display a self-serving bias relative to the model.

Isolating single primary rat hippocampal neurons & astrocytes on ultra-thin patterned parylene-C/silicon dioxide substrates

We report here the patterning of primary rat neurons and astrocytes from the postnatal hippocampus on ultra-thin parylene-C deposited on a silicon dioxide substrate, following observations of neuronal, astrocytic and nuclear coverage on strips of different lengths, widths and thicknesses. Neuronal and glial growth was characterized ‘on’, ‘adjacent to’ and ‘away from’ the parylene strips. In addition, the article reports how the same material combination can be used to isolate single cells along thin tracks of parylene-C. This is demonstrated with a series of high magnification images of the experimental observations for varying parylene strip widths and thicknesses. Thus, the findings demonstrate the possibility to culture cells on ultra-thin layers of parylene-C and localize single cells on thin strips. Such work is of interest and significance to the Neuroengineering and Multi-Electrode Array (MEA) communities, as it provides an alternative insulating material in the fabrication of embedded micro-electrodes, which can be used to facilitate single cell stimulation and recording in capacitive coupling mode.

Twenty-first-century climate impacts from a declining Arctic sea ice cover

A steady decline in Arctic sea ice has been observed over recent decades. General circulation models predict further decreases under increasing greenhouse gas scenarios. Sea ice plays an important role in the climate system in that it influences ocean-to-atmosphere fluxes, surface albedo, and ocean buoyancy. The aim of this study is to isolate the climate impacts of a declining Arctic sea ice cover during the current century. The Hadley Centre Atmospheric Model (HadAM3) is forced with observed sea ice from 1980 to 2000 (obtained from satellite passive microwave radiometer data derived with the Bootstrap algorithm) and predicted sea ice reductions until 2100 under one moderate scenario and one severe scenario of ice decline, with a climatological SST field and increasing SSTs. Significant warming of the Arctic occurs during the twenty-first century (mean increase of between 1.6° and 3.9°C), with positive anomalies of up to 22°C locally. The majority of this is over ocean and limited to high latitudes, in contrast to recent observations of Northern Hemisphere warming. When a climatological SST field is used, statistically significant impacts on climate are only seen in winter, despite prescribing sea ice reductions in all months. When correspondingly increasing SSTs are incorporated, changes in climate are seen in both winter and summer, although the impacts in summer are much smaller. Alterations in atmospheric circulation and precipitation patterns are more widespread than temperature, extending down to midlatitude storm tracks. Results suggest that areas of Arctic land ice may even undergo net accumulation due to increased precipitation that results from loss of sea ice. Intensification of storm tracks implies that parts of Europe may experience higher precipitation rates.

Host model uncertainties in aerosol radiative forcing estimates: results from the AeroCom Prescribed intercomparison study

Simulated multi-model “diversity” in aerosol direct radiative forcing estimates is often perceived as a measure of aerosol uncertainty. However, current models used for aerosol radiative forcing calculations vary considerably in model components relevant for forcing calculations and the associated “host-model uncertainties” are generally convoluted with the actual aerosol uncertainty. In this AeroCom Prescribed intercomparison study we systematically isolate and quantify host model uncertainties on aerosol forcing experiments through prescription of identical aerosol radiative properties in twelve participating models. Even with prescribed aerosol radiative properties, simulated clear-sky and all-sky aerosol radiative forcings show significant diversity. For a purely scattering case with globally constant optical depth of 0.2, the global-mean all-sky top-of-atmosphere radiative forcing is −4.47Wm−2 and the inter-model standard deviation is 0.55Wm−2, corresponding to a relative standard deviation of 12 %. For a case with partially absorbing aerosol with an aerosol optical depth of 0.2 and single scattering albedo of 0.8, the forcing changes to 1.04Wm−2, and the standard deviation increases to 1.01W−2, corresponding to a significant relative standard deviation of 97 %. However, the top-of-atmosphere forcing variability owing to absorption (subtracting the scattering case from the case with scattering and absorption) is low, with absolute (relative) standard deviations of 0.45Wm−2 (8 %) clear-sky and 0.62Wm−2 (11 %) all-sky. Scaling the forcing standard deviation for a purely scattering case to match the sulfate radiative forcing in the Aero- Com Direct Effect experiment demonstrates that host model uncertainties could explain about 36% of the overall sulfate forcing diversity of 0.11Wm−2 in the AeroCom Direct Radiative Effect experiment.

Expression of the guanine nucleotide exchange factor, mr-gef, is regulated during the differentiation of specific subsets of telencephalic neurons

We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.

A systematic approach to identify the sources of tropical SST errors in coupled models using the adjustment of initialised experiments

Understanding the sources of systematic errors in climate models is challenging because of coupled feedbacks and errors compensation. The developing seamless approach proposes that the identification and the correction of short term climate model errors have the potential to improve the modeled climate on longer time scales. In previous studies, initialised atmospheric simulations of a few days have been used to compare fast physics processes (convection, cloud processes) among models. The present study explores how initialised seasonal to decadal hindcasts (re-forecasts) relate transient week-to-month errors of the ocean and atmospheric components to the coupled model long-term pervasive SST errors. A protocol is designed to attribute the SST biases to the source processes. It includes five steps: (1) identify and describe biases in a coupled stabilized simulation, (2) determine the time scale of the advent of the bias and its propagation, (3) find the geographical origin of the bias, (4) evaluate the degree of coupling in the development of the bias, (5) find the field responsible for the bias. This strategy has been implemented with a set of experiments based on the initial adjustment of initialised simulations and exploring various degrees of coupling. In particular, hindcasts give the time scale of biases advent, regionally restored experiments show the geographical origin and ocean-only simulations isolate the field responsible for the bias and evaluate the degree of coupling in the bias development. This strategy is applied to four prominent SST biases of the IPSLCM5A-LR coupled model in the tropical Pacific, that are largely shared by other coupled models, including the Southeast Pacific warm bias and the equatorial cold tongue bias. Using the proposed protocol, we demonstrate that the East Pacific warm bias appears in a few months and is caused by a lack of upwelling due to too weak meridional coastal winds off Peru. The cold equatorial bias, which surprisingly takes 30 years to develop, is the result of an equatorward advection of midlatitude cold SST errors. Despite large development efforts, the current generation of coupled models shows only little improvement. The strategy proposed in this study is a further step to move from the current random ad hoc approach, to a bias-targeted, priority setting, systematic model development approach.