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Strains Splasmids V, vector and primers P used in this study are listed in the supplementary material Table 1S1S2 and S3respectively. All genomic deletions and substitutions were made in ICC strain 1 S1 in Table S2 using a two-step recombination methodology resulting in scarless and markerless mutants as previously described The isogenic strains generated are summarised in Table 1.

Introduction

We generated a vector V9 to insert the P. Transfer of resistance plasmids was achieved by conjugation into KP using an E. Room temperature DH5a S6 competent cells 27 were transformed by electroporation. Capsule production in the isogenic KP strains ICC was measured through quantification of capsular uronic acid as described Isogenic strains were grown rule an OD of 0.

Plates for agar dilution MICs were made in accordance with published guidance 31 using Mueller-Hinton Agar Merck, UK supplemented with meropenem, ertapenem or imipenem. Saturated overnight cultures of test strains were diluted in 0. Proteoliposomes were prepared as previously described with no phale Each curve represents three separate liposome reconstitutions. Plate-like crystals for OmpK36 ST were grown from a solution containing 0. Diffraction screening and data collection were performed at Diamond Light Source synchrotron.

OmpK36 ST data to 3. The space group was determined to be P2 1 2 1 2 with 18 copies of OmpK36 ST in the asymmetric unit. OmpK36 Naked ass cheeks clap data to 1. Data collection statistics are summarised in Table 2. Initial refinement phale OmpK36 ST to 3. After rigid body and restrained refinement extra electron density corresponding to the mutations and insertions were identified, built and refined. Additional density, possibly detergent molecules, that was observed on the surface of the protein was not modelled due to the low resolution.

All model building was performed in Coot Free girls fucking guys with strap statistics are summarised in Table 2. Constriction of the pore diameter was determined by measurement of the channel path by HOLE Anaesthesia was induced by i. This was pipetted into the hub of the cannula and inoculum drawn into the lungs by the inspiratory effort of spontaneously breathing animals.

Fifty microlitres was delivered as outlined above. Images were reconstructed in Living Image 4. For time course experiments mice were housed in groups of 5. Each strain was tested in a total of 10 animals, 5 animals per group in biological duplicate. In each competition assay the tags identifying competing strains were reversed in a biological replicate providing a total number of 10 mice for each competition tested. Inoculum was delivered using the same methodology as for single inoculum infections. Inoculum was enumerated by overnight incubation on LB Agar plates and fluorescent tags identified by blue light transillumination Safe Imager, ThermoFisher Scientific of colonies.

Further information on research design is available in the Nature Research Reporting Summary linked to this article. The source data underlying Figs. Rice, L. Bengoechea, J. Klebsiella pneumoniae infection biology: living to counteract host defences. FEMS Microbiol. Han, J. Epidemiology of carbapenem-resistant Klebsiella pneumoniae in a network of long-term acute care hospitals. Munoz-Price, L.

Phale epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Tacconelli, E. Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. Xu, L. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae.

Mathers, A. The role of epidemic resistance plasmids and international high-risk clones in the spread phale multidrug-resistant Enterobacteriaceae. Pulzova, L. Alterations in outer membrane permeability favor drug-resistant phenotype of Klebsiella pneumoniae. Drug Resist. Peirano, G. Agents Chemother. Nikaido, H. Porins and specific diffusion channels in bacterial outer membranes. Tsai, Y. Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence.

Dutzler, R. Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae. Structure 7— Rule, S. Getting rule into Gram-negative bacteria: rational rules for permeation through general porins.

ACS Infect. Bowers, J. Genomic analysis of the emergence and rapid global dissemination of the clonal group Klebsiella pneumoniae pandemic. PLoS One 10e Rule, C. Mutations of the ompK36 porin gene and promoter impact responses of sequence typeKPCproducing Klebsiella pneumoniae strains to doripenem and doripenem-colistin. Agyekum, A.

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Huang, W. In-hospital medical costs of infections caused by carbapenem-resistant Klebsiella pneumoniae. Pitout, J. Carbapenemase-producing Klebsiella pneumoniaea key pathogen set for global nosocomial dominance. Humphries, R. Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC Bbw cheerleader, R. Ventilator-associated pneumonia: the central role of transcolonization. Care 50— Mitra, R.

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Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7. DNA 118 Paczosa, M. Klebsiella pneumoniae : going on the offense with a strong defense. Klemm, E. Emergence of dominant multidrug-resistant bacterial clades: Lessons from history and whole-genome sequencing. Natl Acad. USA— Public Health England.

Berger, C.

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Citrobacter rodentium subverts ATP flux and cholesterol homeostasis in intestinal epithelial cells in vivo. Cell Metab. Tu, Q. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency. Dorman, M. MBio 9e Wise, M. Global survey of Klebsiella pneumoniae major porins from ertapenem non-susceptible isolates lacking carbapenemases. Breakpoint tables for interpretation of MICs and zone diameters. Prilipov, A.

Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. Beis, K. Two-step purification of outer membrane proteins. Porin channels in Escherichia coli : studies with liposomes reconstituted from purified proteins.

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Vonrhein, C. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Winter, G. McCoy, A. Phaser crystallographic software. Murshudov, G. Refinement of macromolecular structures by the maximum-likelihood method. Adams, P. Emsley, P. Coot: Model-building tools for molecular graphics. Smart, O. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. Download references.

All animal work described in this study was funded by grants from the Medical Research Council and the Wellcome Trust. They analysed data. SJB provided clinical input and edited the paper.

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Correspondence to Gad Frankel. Peer review information: Nature Communications thanks Susan K. Buchanan and the other, anonymous, reviewer s for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and Permissions. Wong, J. Nat Commun 10, doi Download citation.

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