Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.     

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.     

Characterization and application of a vinblastine-selected CACO-2 cell line for evaluation of p-glycoprotein

The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.     

Rapid tightening of blood glucose control leads to transient deterioration of retinopathy in insulin dependent diabetes mellitus: the Oslo study.

In a study of retinopathy during one year of tight blood glucose control 45 type I (insulin dependent) diabetics without proliferative retinopathy were randomised to receive either continuous subcutaneous insulin infusion, multiple insulin injections, or conventional insulin treatment (controls). Near normoglycaemia was achieved with continuous infusion and multiple injections but not with conventional treatment. Blind evaluation of fluorescein angiograms performed three monthly showed progression of retinopathy in the control group, transient deterioration in the continuous infusion group, and no change in the multiple injection group. Half the patients receiving continuous infusion and multiple injections developed retinal cotton wool spots after three to six months. These changes regressed in all but four patients after 12 months. Control patients did not develop cotton wool spots. Patients who developed cotton wool spots are characterised by a larger decrement in glycosylated haemoglobin and blood glucose values, more frequent episodes of hypoglycaemia, a longer duration of diabetes, and more severe retinopathy at onset. A large and rapid fall in blood glucose concentration may promote transient deterioration of diabetic retinopathy.     

Breaking the ice: large-scale distribution of mesozooplankton after a decade of Arctic and transpolar cruises

Mesozooplankton collected during five summer expeditions to the Arctic Ocean between 1987 and 1991 was analysed for regional patterns in biomass and species distribution, distinguishing between an epipelagic (0–100 m) and a deeper (0–500 m) layer. A total of 58 stations was sampled mainly in the Nansen, Amundsen and Makarov Basins of the central Arctic Ocean and in areas of the Greenland Sea, West Spitsbergen Current and Barents Sea. Results from the different expeditions were combined to create a transect extending from the Fram Strait across the Eurasian Basin into the Makarov Basin. Mesozooplankton dry mass in the upper 500 m decreased from 8.4 g m⁻² in the West Spitsbergen Current to less than 2 g m⁻² in the high-Arctic deep-sea basins. In the central Arctic Ocean, biomass was concentrated in the upper 100 m and was dominated by the large copepods Calanus hyperboreus and C. glacialis. In contrast, the mesozooplankton in the West Spitsbergen Current was more evenly distributed throughout the upper 500 m, with C. finmarchicus as the prevailing species. The distribution of abundant mesopelagic species reflected the hydrographic regime: the calanoid copepod Gaetanus tenuispinus and the hyperiid amphipod Themisto abyssorum were most abundant in the Atlantic inflow, while Scaphocalanus magnus was a typical component of the high-Arctic fauna. The relatively high mesozooplankton biomass and the occurrence of boreal-Atlantic species in the central Arctic Ocean are indicators for the import of organic material from allochthonous sources, especially from the northern North Atlantic. Hence, in spite of its enclosure by land masses, the Arctic Ocean is characterized by an exchange of water masses and organisms with the North Atlantic, and advection processes strongly influence the distribution of plankton species in this high-latitude ecosystem. Received: 18 December 1997 / Accepted: 11 April 1998     

Long-term Infusion of Growth Hormone Release Inhibiting Hormone in Acromegaly: Effects on Pituitary and Pancreatic Hormones

Growth hormone release inhibiting hormone (GH-RIH) was infused at a rate of 1·3 μg/min for 28 hours into four patients with acromegaly, two of whom also had clinical diabetes mellitus. Growth hormone and glucagon were suppressed throughout the infusion though delayed secretion of insulin occurred in association with both meals and an oral glucose load. Glucose tolerance was improved in one diabetic patient who was taking chlorpropamide while the other required much less insulin than usual. Secretion of endogenous thyroid-stimulating hormone was lowered in one euthyroid patient on carbimazole. Luteinizing hormone, follicle-stimulating hormone, ACTH, and prolactin were not affected. Serum somatomedin levels were reduced in one patient. There was a rapid rebound of all the suppressed hormones when the infusions stopped. Longer-acting analogues of GH-RIH will be needed before long-term therapy of acromegaly or diabetes mellitus becomes possible, but such preparations should be available soon for clinical trial.     

A Look into the Melting Pot: The mecC-Harboring Region Is a Recombination Hot Spot in Staphylococcus stepanovicii

Introduction

Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus.

Methods

Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on “Rodent-Borne Pathogens”) in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KR732654","term_id":"922979931"}}KR732654 and {"type":"entrez-nucleotide","attrs":{"text":"KR732653","term_id":"922979910"}}KR732653).

Results

The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal “hot spot” for foreign DNA integration and exchange.

Conclusion

Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.     

The environmental effectiveness of the beverage sector in Norway in a Factor 10 perspective

Scope and Background

The environmental effectiveness of the Norwegian beverage sector has been studied in a Factor 10 perspective. The objective of the study was to identify strategies that could make the beverage sector radically more effective from an environmental and resource perspective, leading to a Factor 10 improvement. Another main purpose of the work was to test the potential for using Life Cycle Assessment (LCA) methodology on an economic sector with a network of product chains, rather than for a single product.

Methods

Life Cycle Assessment data from STØ’s own studies and literature studies have been used as a basis for analysis of the environmental status of the beverage sector in Norway. The functional unit was defined as the amount of beverage products consumed per capita in Norway in the year 2000. The study includes raw material production, production of the beverage product, packaging manufacture, distribution, use and waste management of the products. The study has, for practical reasons, been limited to the environmental impact indicators total energy consumption and global warming potential. This was done as other types of data have been difficult to obtain for all of the products that were studied (tap water, coffee, milk, soft drinks, beer, squash, juice and bottled water).

Results and Discussion

The study shows differences between the drinking products with respect to energy consumption and emissions that can contribute to global warming. Due to large uncertainties in the data, general conclusions regarding the differentiation of products based on environmental performance should be made with care. Production and distribution of tap water is, however, significantly less energy intensive than the other products. For the impact categories studied, production of raw materials was the most important part of the life cycle for most drinking products.

Conclusions and Perspective

The most significant contributions to achieving a Factor 10 development can be made by consuming more water, especially tap water, and through improving raw material production in the agricultural sector. Packaging and distribution is responsible for only a small part of the energy consumption and emissions leading to global warming. Optimal packaging sizes might however reduce loss of products in the user phase, which is important in order to improve the system. A Factor 10 level seems achievable only if the consumption of tap water is increased to a high level.