Comparison of a monoclonal antibody-based capture/enrichment sandwich enzyme linked immunosorbent assay with immunomagnetic bead separation for the detection of attachment effacement Escherichia coli O26 strains from cattle faeces

AIMS: A monoclonal antibody (Mab 2F3)-based sandwich enzyme linked immunosorbent assay (sELISA) format for the detection of Escherichia coli O26 that improves the sensitivity of the assay by combining enrichment with the capture stage has been developed. Culture of the enriched contents of wells before completion of the sELISA was compared with immunomagnetic bead separation (IMS) as a means of specific isolation of the target organism. METHODS AND RESULTS: Bovine faecal samples, c. 10% in buffered peptone water (BPW), were pre-enriched for 6 h before testing by capture/enrichment sELISA and by IMS. The sELISA consisted of a 1-2 h capture stage followed by addition of BPW to the wells and an overnight enrichment stage before completion of the assay. The capture/enrichment stage of the assay was repeated a second time on the enriched contents removed from the wells before completion of the first sELISA. From 204 cattle faeces samples, 30x O26 strains [20x attachment effacement Escherichia coli (AEEC) and 10x non-AEEC] were isolated from the enriched wells of the sELISA, in comparison with 11 (9x AEEC and 2x non-AEEC) that were isolated by IMS. Examination of the use of enterohaemolysin activity and rhamnose utilization on 1% rhamnose McConkey's (RMAC) agar with or without cefixime and potassium tellurite demonstrated that the selection based on enterohaemolysin production and growth on RMAC with cefixime and potassium tellurite would largely differentiate the AEEC strains from the non-AEEC strains. CONCLUSIONS: The capture/enrichment sELISA protocol used compared favourably with the IMS for the isolation of E. coli O26 from faeces samples. The ELISA optical density readings obtained in the procedure were used as a screening indicator for selection of samples for further culture examination, and the selective culture methods examined to assist strain isolation did have potential. SIGNIFICANCE AND IMPACT OF THE STUDY: The capture/enrichment format of an Mab-based sELISA protocol has the potential to provide a suitable screening assay for the specific detection of pathogenic strains from mixed culture samples like faeces.     

Multiple cosmopolitan ecotypes within a microbial eukaryote morphospecies

Microbial eukaryotes that are morphologically indistinguishable (i.e. 'morphospecies') tend to be genetically diverse. While most protist morphospecies have cosmopolitan distribution, it has been suggested that ribotypes (unique rRNA gene sequences) or rRNA sequence clusters do have biogeography and such clusters may correlate with particular (non-morphological) adaptations. We have studied this in the ciliated protozoan morphospecies Cyclidium glaucoma. Fifty-four isolates collected worldwide represented 31 distinct ribotypes. There was no evidence of biogeographic distribution patterns. For example, identical ribotypes occurred in samples from Argentina, Peru, Morocco, Russia and Ukraine; in samples from Denmark and Australia; and in samples from Great Salt Lake and hyperhaline ponds in Spain. The morphospecies Cyclidium glaucoma is euryhaline and occurs in freshwater, brackish water, seawater, and hyperhaline waters. Evidence suggests that one ribotype cluster occurs only in marine or brackish habitats, and another one has so far been found only in hyperhaline habitats. Two clades seem to occur only in freshwater, but one clade includes ribotypes that were found in freshwater as well as in brackish water.     

Bacterial microbiomes of individual ectomycorrhizal Pinus sylvestris roots are shaped by soil horizon and differentially sensitive to nitrogen addition

Plant roots select non‐random communities of fungi and bacteria from the surrounding soil that have effects on their health and growth, but we know little about the factors influencing their composition. We profiled bacterial microbiomes associated with individual ectomycorrhizal Pinus sylvestris roots colonized by different fungi and analyzed differences in microbiome structure related to soils from distinct podzol horizons and effects of short‐term additions of N, a growth‐limiting nutrient commonly applied as a fertilizer, but known to influence patterns of carbon allocation to roots. Ectomycorrhizal roots growing in soil from different horizons harboured distinct bacterial communities. The fungi colonizing individual roots had a strong effect on the associated bacterial communities. Even closely related species within the same ectomycorrhizal genus had distinct bacterial microbiomes in unfertilized soil, but fertilization removed this specificity. Effects of N were rapid and context dependent, being influenced by both soil type and the particular ectomycorrhizal fungi involved. Fungal community composition changed in soil from all horizons, but bacteria only responded strongly to N in soil from the B horizon where community structure was different and bacterial diversity was significantly reduced, possibly reflecting changed carbon allocation patterns.     

Marine anti-biofouling efficacy of amphiphilic poly(coacrylate) grafted PDMSe: effect of graft molecular weight

There is currently strong motivation due to ecological concerns to develop effective anti-biofouling coatings that are environmentally benign, durable, and stable for use by the maritime industry. The antifouling (AF) and fouling-release (FR) efficacy of amphiphilic, charged copolymers composed of ~52% acrylamide, ~34% acrylic acid, and ~14% methyl acrylate grafted to poly(dimethyl siloxane) (PDMSe) surfaces were tested against zoospores of the green alga Ulva linza and the diatom Navicula incerta. The biofouling response to molecular weight variation was analyzed for grafts ranging from ~100 to 1,400 kg mol^?1, The amphiphilic coatings showed a marked improvement in the FR response, with a 55% increase in the percentage removal of diatoms and increased AF efficacy, with 92% reduction in initial attachment density of zoospores, compared to PDMSe controls. However, graft molecular weight, in the range tested, was statistically insignificant. Grafting copolymers to PDMSe embossed with the Sharklet? microtopography did not produce enhanced AF efficacy.     

Impact of physician continuity on death or urgent readmission after discharge among patients with heart failure

Background:

Early physician follow-up after discharge is associated with lower rates of death and readmission among patients with heart failure. We explored whether physician continuity further influences outcomes after discharge.

Methods:

We used data from linked administrative databases for all adults aged 20 years or more in the province of Alberta who were discharged alive from hospital between January 1999 and June 2009 with a first-time diagnosis of heart failure. We used Cox proportional hazard models with time-dependent covariates to analyze the effect of follow-up with a familiar physician within the first month after discharge on the primary outcome of death or urgent all-cause readmission over 6 months. A familiar physician was defined as one who had seen the patient at least twice in the year before the index admission or once during the index admission.

Results:

In the first month after discharge, 5336 (21.9%) of the 24 373 identified patients had no follow-up visits, 16 855 (69.2%) saw a familiar physician, and 2182 (9.0%) saw unfamiliar physician(s) exclusively. The risk of death or unplanned readmission during the 6-month observation period was lower among patients who saw a familiar physician (43.6%; adjusted hazard ratio [HR] 0.87, 95% confidence interval [CI] 0.83–0.91) or an unfamiliar physician (43.6%; adjusted HR 0.90, 95% CI 0.83–0.97) for early follow-up visits, as compared with patients who had no follow-up visits (62.9%). Taking into account all follow-up visits over the 6-month period, we found that the risk of death or urgent readmission was lower among patients who had all of their visits with a familiar physician than among those followed by unfamiliar physicians (adjusted HR 0.91, 95% CI 0.85–0.98).

Interpretation:

Early physician follow-up after discharge and physician continuity were both associated with better outcomes among patients with heart failure. Research is needed to explore whether physician continuity is important for other conditions and in settings other than recent hospital discharge.Hospital care accounts for almost one-third of health care spending, and unplanned readmissions within 30 days after discharge cost more than $20 billion each year in the United States and Canada.¹ Heart failure is one of the most common reasons for admission to hospital and is associated with a high risk of readmission.¹ Although the prognosis for patients with heart failure has improved over the past decade, the risk of early death or readmission after discharge is still high and is increasing.² Prompt follow-up of patients with heart failure has been associated with lower rates of death and readmission,^3,4 and 30-day follow-up has been included as a quality-of-care indicator in Canada.⁵It is unclear, however, whether the postdischarge visits should be with the physician who previously saw the patient or with any physician. Results of studies exploring the association between provider continuity and postdischarge outcomes have been inconclusive and the studies have included few patients with heart failure.^6–9 Intuitively, one might consider physician continuity important for patients with heart failure discharged from hospital, given their age, high comorbidity burdens and complex treatment regimens. However, a robust evidence base and multiple guidelines with consistent messaging on key management principles have made physician continuity potentially less important.We designed this study to determine whether physician continuity influenced postdischarge outcomes among patients with heart failure beyond the influence of early physician follow-up.     

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70–80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r ² > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.     

The role of surface energy and water wettability in aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel surfaces in the control of marine biofouling

Xerogel films with uniform surface topogrophy, as determined by scanning electron microscopy, atomic force microscopy (AFM), and time-of-flight secondary ion mass spectrometry, were prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl- containing precursors. Young's modulus was determined from AFM indentation measurements. The xerogel coatings gave reduced settlement of zoospores of the marine fouling alga Ulva compared to a poly(dimethylsiloxane) elastomer (PDMSE) standard. Increased settlement correlated with decreased water wettability as measured by the static water contact angle, θ_Ws, or with decreased polar contribution (γ_P) to the surface free energy (γ_S) as measured by comprehensive contact angle analysis. The strength of attachment of 7-day sporelings (young plants) of Ulva on several of the xerogels was similar to that on PDMSE although no overall correlation was observed with either θ_Ws or γ_S. For sporelings attached to the fluorocarbon/hydrocarbon-modified xerogels, the strength of attachment increased with increased water wettability. The aminopropyl-modified xerogels did not follow this trend.     

Computer-based assessment of movement difficulties in Parkinson's disease

The prevalence of Parkinson's disease (PD) is increasing due to an ageing population. It is an unpredictable disease which requires regular assessment and monitoring. Current techniques used to assess PD are subjective. Clinicians observe movements made by a patient and subsequently rate the level of severity of, for example tremor or slowness of movement. Within this work, we have developed and evaluated a prototype computer-based assessment tool capable of collecting information on the movement difficulties present in PD. Twenty participants took part in an assessment of the tool, 10 of whom were diagnosed with PD and 10 were without the disease. Following the usage of the tool, it was found that there was a significant difference (p = 0.038) in the speed of movement between the two groups. We envisage that this tool could have the potential to enable more objective clinical conclusions to be made.     

Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases.     

Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase

Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.