Complete Nucleotide Sequence of CTX-M-15-Plasmids from Clinical Escherichia coli Isolates: Insertional Events of Transposons and Insertion Sequences

Background

CTX-M-producing Escherichia coli strains are regarded as major global pathogens.

Methodology/Principal Findings

The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla _TEM-1 and bla _CTX-M-15. It shares more than 90% homology with a previously published bla _CTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla _TEM-1 and bla _CTX-M-15, were found. Six resistance genes, bla _TEM-1, bla _CTX-M-15, bla _OXA-1, aac6''-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla _CTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla _TEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla _OXA-1, aac6''-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids.

Conclusions

Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.     

Angiopoietin-1 treatment reduces inflammation but does not prevent ventilator-induced lung injury

Background

Loss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie2 system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie2 system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie2 receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation.

Methods

Mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH₂O (‘low’ tidal volume ∼7.5 ml/kg; LV_T) or 18 cmH₂O (‘high’ tidal volume ∼15 ml/kg; HV_T). At initiation of HV_T-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls.

Results

HV_T-ventilation influenced the Ang-Tie2 system in lungs of healthy mice since Ang-1, Ang-2 and Tie2 mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie2 signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HV_T-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HV_T-ventilated mice despite inhibition of inflammation, vascular endothelial growth factor (VEGF) and Ang-2 expression.

Conclusions

Ang-1 treatment downregulates pulmonary inflammation, VEGF and Ang-2 expression but does not protect against vascular leakage and impaired gas exchange induced by HV_T-ventilation.     

Initial decay of woody fragments in soil is influenced by size, vertical position, nitrogen availability and soil origin

Fast-growing bacteria and fungi are expected to cause the initial stage of decomposition of woody fragments in and on soils, i.e. the respiration of sugars, organic acids, pectin and easily accessible cellulose and hemi-cellulose. However, little is known about the factors regulating initial wood decomposition. We examined the effect of wood fragment size, vertical position, nitrogen addition and soil origin on initial wood decay and on the relative importance of fungi and bacteria therein. Two fractions of birch wood were used in microcosm experiments, namely wood blocks (dimensions: 3 × 0.5 × 0.5 cm) and sawdust (dimensions: 0.5–2 mm). The woody fragments were enclosed in nylon bags and placed on top of- or buried in an abandoned arable soil and in a heathland soil. After 15, 25 and 40 weeks of incubation, fungal biomass was quantified (as ergosterol and chitin content) and bacterial numbers were determined. The results indicated that initial wood decay was mostly caused by fungi; bacteria were only contributing in sawdust in/on abandoned arable soil. Larger fragment size, burial of fragments and nitrogen addition positively influenced fungal biomass and activity. Fungal biomass and decay activities were much lower in woody fragments incubated in/on heathland soil than in those incubated in/on abandoned arable soil, indicating that soil origin is also an important factor determining initial wood decay.     

The role of lipids in membrane insertion and translocation of bacterial proteins

Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2)

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.     

Vitamin D(3): synthesis of seco C-9,11,21-trisnor-17-methyl-1 alpha, 25-dihydroxyvitamin D(3) analogues

The synthesis and biological activities of seco C-9,11,21-trisnor-17-methyl-1 alpha,25-dihydroxyvitamin D(3) analogues (D-ring analogues) are described.