Evidence for selection of multi-resistant E. coli by hospital effluent.

There is a risk that residues of antibiotics and other antimicrobials in hospital and municipal wastewaters could select for resistant bacteria. Still, direct experimental evidence for selection is lacking. Here, we investigated if effluent from a large Swedish hospital, as well as influent and effluent from the connected municipal wastewater treatment plant (WWTP) select for antibiotic resistant Escherichia coli in three controlled experimental setups. Exposure of sterile-filtered hospital effluent to a planktonic mix of 149 different E. coli wastewater isolates showed a strong selection of multi-resistant strains. Accordingly, exposure to a complex wastewater community selected for strains resistant to several antibiotic classes. Exposing individual strains with variable resistance patterns revealed a rapid bactericidal effect of hospital effluent on susceptible, but not multi-resistant E. coli. No selection was observed after exposure to WWTP effluent, while exposure to WWTP influent indicated a small selective effect for ceftazidime and cefadroxil resistant strains, and only in the E. coli mix assay. An analysis of commonly used antibiotics and non-antibiotic pharmaceuticals in combination with growth and resistance pattern of individual E. coli isolates suggested a possible contribution of ciprofloxacin and ß-lactams to the selection by hospital effluent. However, more research is needed to clarify the contribution from different selective agents. While this study does not indicate selection by the studied WWTP effluent, there is some indications of selective effects by municipal influent on ß-lactam-resistant strains. Such effects may be more pronounced in countries with higher antibiotic use than Sweden. Despite the limited antibiotic use in Sweden, the hospital effluent strongly and consistently selected for multi-resistance, indicating widespread risks. Hence, there is an urgent need for further evaluation of risks for resistance selection in hospital sewers, as well as for strategies to remove selective agents and resistant bacteria.

Synthesis and biological evaluation of truncated derivatives of abyssomicin C as antibacterial agents.

The synthesis and antibacterial activity of two new highly truncated derivatives of the natural product abyssomicin C are reported. This work outlines the limits of structural truncation of the natural product and consequently provides insights for further structure-activity relationship studies towards novel antibiotics targeting 4-amino-4-deoxychorismate (ADC) synthase. Specifically, it is demonstrated that the synthetically challenging bicyclic motif is essential for activity towards methicillin-resistant Staphylococcus aureus (MRSA).

A Comprehensive Screening of Escherichia coli Isolates from Scandinavia's Largest Sewage Treatment Plant Indicates No Selection for Antibiotic Resistance.

There is concern that sewage treatment plants (STPs) serve as hotspots for emergence and selection of antibiotic resistant bacteria. However, field studies investigating resistance selection by comparing bacterial populations in influents and effluents have produced variable and sometimes contradictive results. Also, large taxonomic changes between influents and effluents make interpretation of studies measuring relative gene abundances ambiguous. The aim here was to investigate whether within-species selection occurs by conducting a comprehensive screening of Escherichia coli isolated from composite influent and effluent samples collected at Scandinavia's largest STP, accompanied by analyses of antibiotics residues. In total, 4028 isolates, collected on eight occasions during 18 months, were screened for resistance to seven antibiotics. Although differences in proportions of resistant E. coli between influent and effluent samples were detected for a few antibiotics on two occasions, aggregated data over time showed no such differences for any of the investigated antibiotics. Neither was there any enrichment of multiresistant or extended-spectrum beta-lactamase-producing isolates through the treatment process. Despite some antibiotics were detected at or close to concentrations predicted to provide some selective pressure, field observations of resistance profiles in E. coli do not provide support for systematic selection in the investigated STP.

Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

Dynamics of elongation factor 2 kinase regulation in cortical neurons in response to synaptic activity.

The rapid regulation of cell signaling in response to calcium in neurons is essential for real-time processing of large amounts of information in the brain. A vital regulatory component, and one of the most energy-intensive biochemical processes in cells, is the elongation phase of mRNA translation, which is controlled by the Ca(2+)/CaM-dependent elongation factor 2 kinase (eEF2K). However, little is known about the dynamics of eEF2K regulation in neurons despite its established role in learning and synaptic plasticity. To explore eEF2K dynamics in depth, we stimulated synaptic activity in mouse primary cortical neurons. We find that synaptic activity results in a rapid, but transient, increase in eEF2K activity that is regulated by a combination of AMPA and NMDA-type glutamate receptors and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways. We then used computational modeling to test the hypothesis that considering Ca(2+)-coordinated MEK/ERK, mTORC1, and eEF2k activation is sufficient to describe the observed eEF2K dynamics. Although such a model could partially fit the empirical findings, it also suggested that a crucial positive regulator of eEF2K was also necessary. Through additional modeling and empirical evidence, we demonstrate that AMP kinase (AMPK) is also an important regulator of synaptic activity-driven eEF2K dynamics in neurons. Our combined modeling and experimental findings provide the first evidence that it is necessary to consider the combined interactions of Ca(2+) with MEK/ERK, mTORC1, and AMPK to adequately explain eEF2K regulation in neurons.

BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.