Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

Background and aim: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between ampli-cons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing. © 2018, European Centre for Disease Prevention and Control (ECDC). All rights reserved

The increasing importance of atmospheric demand for ecosystem water and carbon fluxes

Soil moisture supply and atmospheric demand for water independently limit—and profoundly affect—vegetation productivity and water use during periods of hydrologic stress1, 2, 3, 4. Disentangling the impact of these two drivers on ecosystem carbon and water cycling is difficult because they are often correlated, and experimental tools for manipulating atmospheric demand in the field are lacking. Consequently, the role of atmospheric demand is often not adequately factored into experiments or represented in models5, 6, 7. Here we show that atmospheric demand limits surface conductance and evapotranspiration to a greater extent than soil moisture in many biomes, including mesic forests that are of particular importance to the terrestrial carbon sink8, 9. Further, using projections from ten general circulation models, we show that climate change will increase the importance of atmospheric constraints to carbon and water fluxes in all ecosystems. Consequently, atmospheric demand will become increasingly important for vegetation function, accounting for >70% of growing season limitation to surface conductance in mesic temperate forests. Our results suggest that failure to consider the limiting role of atmospheric demand in experimental designs, simulation models and land management strategies will lead to incorrect projections of ecosystem responses to future climate conditions

Signs and symptoms in children with a serious infection: a qualitative study

BACKGROUND: Early diagnosis of serious infections in children is difficult in general practice, as incidence is low, patients present themselves at an early stage of the disease and diagnostic tools are limited to signs and symptoms from observation, clinical history and physical examination. Little is known which signs and symptoms are important in general practice. With this qualitative study, we aimed to identify possible new important diagnostic variables. METHODS: Semi-structured interviews with parents and physicians of children with a serious infection. We investigated all signs and symptoms that were related to or preceded the diagnosis. The analysis was done according to the grounded theory approach. Participants were recruited in general practice and at the hospital. RESULTS: 18 children who were hospitalised because of a serious infection were included. On average, parents and paediatricians were interviewed 3 days after admittance of the child to hospital, general practitioners between 5 and 8 days after the initial contact. The most prominent diagnostic signs in seriously ill children were changed behaviour, crying characteristics and the parents' opinion. Children either behaved drowsy or irritable and cried differently, either moaning or an inconsolable, loud crying. The parents found this illness different from previous illnesses, because of the seriousness or duration of the symptoms, or the occurrence of a critical incident. Classical signs, like high fever, petechiae or abnormalities at auscultation were helpful for the diagnosis when they were present, but not helpful when they were absent. CONCLUSION: behavioural signs and symptoms were very prominent in children with a serious infection. They will be further assessed for diagnostic accuracy in a subsequent, quantitative diagnostic study

Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

<p>Abstract</p> <p>Background</p> <p>Plasmid encoded ^<it>bla</it>CTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in <it>Enterobacteriaceae </it>including <it>Klebsiella </it>spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.</p> <p>Methods</p> <p>Multiple displacement amplified DNA derived from 20 <it>K. pneumoniae </it>and 34 <it>K. oxytoca </it>clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.</p> <p>Results</p> <p>Nine out of 20 <it>K. pneumoniae </it>clinical isolates had a ^<it>bla</it>CTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 <it>K. oxytoca </it>clinical isolates. Molecular identification and differentiation between ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. <it>In silico </it>analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located ^<it>bla</it>OXY and K1-genes in <it>Klebsiella </it>spp. and K1-like genes in other <it>Enterobacteriaceae</it>.</p> <p>Conclusion</p> <p>The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of ^<it>bla</it>CTX-M, and ^<it>bla</it>OXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.</p

Search for new physics with same-sign isolated dilepton events with jets and missing transverse energy

A search for new physics is performed in events with two same-sign isolated leptons, hadronic jets, and missing transverse energy in the final state. The analysis is based on a data sample corresponding to an integrated luminosity of 4.98 inverse femtobarns produced in pp collisions at a center-of-mass energy of 7 TeV collected by the CMS experiment at the LHC. This constitutes a factor of 140 increase in integrated luminosity over previously published results. The observed yields agree with the standard model predictions and thus no evidence for new physics is found. The observations are used to set upper limits on possible new physics contributions and to constrain supersymmetric models. To facilitate the interpretation of the data in a broader range of new physics scenarios, information on the event selection, detector response, and efficiencies is provided.Comment: Published in Physical Review Letter

Constraints on the χ_(c1) versus χ_(c2) polarizations in proton-proton collisions at √s = 8 TeV

The polarizations of promptly produced χ_(c1) and χ_(c2) mesons are studied using data collected by the CMS experiment at the LHC, in proton-proton collisions at √s=8  TeV. The χ_c states are reconstructed via their radiative decays χ_c → J/ψγ, with the photons being measured through conversions to e⁺e⁻, which allows the two states to be well resolved. The polarizations are measured in the helicity frame, through the analysis of the χ_(c2) to χ_(c1) yield ratio as a function of the polar or azimuthal angle of the positive muon emitted in the J/ψ → μ⁺μ⁻ decay, in three bins of J/ψ transverse momentum. While no differences are seen between the two states in terms of azimuthal decay angle distributions, they are observed to have significantly different polar anisotropies. The measurement favors a scenario where at least one of the two states is strongly polarized along the helicity quantization axis, in agreement with nonrelativistic quantum chromodynamics predictions. This is the first measurement of significantly polarized quarkonia produced at high transverse momentum

Combined search for the quarks of a sequential fourth generation

Results are presented from a search for a fourth generation of quarks produced singly or in pairs in a data set corresponding to an integrated luminosity of 5 inverse femtobarns recorded by the CMS experiment at the LHC in 2011. A novel strategy has been developed for a combined search for quarks of the up and down type in decay channels with at least one isolated muon or electron. Limits on the mass of the fourth-generation quarks and the relevant Cabibbo-Kobayashi-Maskawa matrix elements are derived in the context of a simple extension of the standard model with a sequential fourth generation of fermions. The existence of mass-degenerate fourth-generation quarks with masses below 685 GeV is excluded at 95% confidence level for minimal off-diagonal mixing between the third- and the fourth-generation quarks. With a mass difference of 25 GeV between the quark masses, the obtained limit on the masses of the fourth-generation quarks shifts by about +/- 20 GeV. These results significantly reduce the allowed parameter space for a fourth generation of fermions.Comment: Replaced with published version. Added journal reference and DO