Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

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Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.publishedVersio

Knockout of Targeted Plasmid-Borne β-Lactamase Genes in an Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile

Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne β-lactamase genes (blaOXA-1, blaTEM-1, and blaCTX-M15) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted β-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the blaCTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of blaOXA-1 and blaTEM-1 affected fewer proteins’ expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss.publishedVersio

Scandinavium goeteborgense gen. nov., sp. nov., a New Member of the Family Enterobacteriaceae Isolated From a Wound Infection, Carries a Novel Quinolone Resistance Gene Variant

The family Enterobacteriaceae is a taxonomically diverse and widely distributed family containing many human commensal and pathogenic species that are known to carry transferable antibiotic resistance determinants. Characterization of novel taxa within this family is of great importance in order to understand the associated health risk and provide better treatment options. The aim of the present study was to characterize a Gram-negative bacterial strain (CCUG 66741) belonging to the family Enterobacteriaceae, isolated from a wound infection of an adult patient, in Sweden. Initial phenotypic and genotypic analyses identified the strain as a member of the family Enterobacteriaceae but could not assign it to any previously described species. The complete 16S rRNA gene sequence showed highest similarity (98.8%) to four species. Whole genome sequencing followed by in silico DNA-DNA similarity analysis and average nucleotide identity (ANI) analysis confirmed that strain CCUG 66741 represents a novel taxon. Sequence comparisons of six house-keeping genes (16S rRNA, atpD, dnaJ, gyrB, infB, rpoB) with those of the type strains of the type species of related genera within the family Enterobacteriaceae indicated that the strain embodies a novel species within the family. Phylogenomic analyses (ANI-based and core genome-based phylogeny) showed that strain CCUG 66741 forms a distinct clade, representing a novel species of a distinct, new genus within the family Enterobacteriaceae, for which the name Scandinavium goeteborgense gen. nov., sp. nov. is proposed, with CCUG 66741T as the type strain (= CECT 9823T = NCTC 14286T). S. goeteborgense CCUG 66741T carries a novel variant of a chromosomally-encoded quinolone resistance gene (proposed qnrB96). When expressed in Escherichia coli, the qnrB96 gene conferred five-fold increase in minimum inhibitory concentration against ciprofloxacin. This study highlights the importance and the utility of whole genome sequencing for pathogen identification in clinical settings.publishedVersio

Seawater from Bergen harbor is a reservoir of conjugative multidrug-resistance plasmids carrying genes for virulence

Aquatic environments play important roles in the dissemination of clinically-relevant antibiotic resistance genes (ARGs) and pathogens. Limited knowledge exists about the prevalence of clinically-relevant acquired resistance genes in the marine environment, especially in Norway. The aim of the current study was to investigate the presence of and characterize self-transmissible resistance plasmids from Bergen harbor seawater, with exogenous-plasmid capture, using a green fluorescent protein (GFP)-tagged Escherichia coli strain as a recipient. We obtained transconjugants resistant against ampicillin and cefotaxime from four of the 13 samples processed. Nine transconjugants, selected on the basis of antibiotic sensitivity patterns, were sequenced, using Illumina MiSeq and Oxford Nanopore MinION platforms. Ten different plasmids (ranging from 35 kb to 136 kb) belonging to incompatibility groups IncFII/IncFIB/Col156, IncFII, IncI1 and IncB/O/K/Z were detected among these transconjugants. Plasmid p1A1 (IncFII/IncFIB/Col156, 135.7 kb) carried resistance genes blaTEM-1, dfrA17, sul1, sul2, tet(A), mph(A), aadA5, aph(3″)-Ib and aph(6)-Id, conferring resistance against six different classes of antibiotics. Plasmid p1A4 carried blaCTX-M-55, lnu(F), aadA17 and aac(3)-IId. Cephalosporinase blaCMY-2 was detected on plasmids captured from an area impacted by wastewater from a local marine aquarium. Along with ARGs, some plasmids also carried virulence factors, such as enterotoxins, adhesion factors and siderophores. Our study demonstrates the presence of clinically-important multidrug-resistance conjugative plasmids in seawater from Bergen harbor, which have the potential to be transferred to human microbiota. The results highlight the need for surveillance of antibiotic resistance in the environment, as suggested by the World Health Organization, especially in low prevalence settings like Norway.publishedVersio

Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

RATIONALE: Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. OBJECTIVES: Extending this work to beta 2 adrenergic systems in vitro and in vivo. METHODS: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. MEASUREMENTS: Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. MAIN RESULTS: Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3-10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in "asthmatic" sheep. CONCLUSIONS: Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications

Implementation salvage experiences from the Melbourne diabetes prevention study

Background Many public health interventions based on apparently sound evidence from randomised controlled trials encounter difficulties when being scaled up within health systems. Even under the best of circumstances, implementation is exceedingly difficult. In this paper we will describe the implementation salvage experiences from the Melbourne Diabetes Prevention Study, which is a randomised controlled trial of the effectiveness and cost-effectiveness nested in the state-wide Life! Taking Action on Diabetes program in Victoria, Australia.Discussion The Melbourne Diabetes Prevention Study sits within an evolving larger scale implementation project, the Life! program. Changes that occurred during the roll-out of that program had a direct impact on the process of conducting this trial. The issues and methods of recovery the study team encountered were conceptualised using an implementation salvage strategies framework. The specific issues the study team came across included continuity of the state funding for Life! program and structural changes to the Life! program which consisted of adjustments to eligibility criteria, referral processes, structure and content, as well as alternative program delivery for different population groups. Staff turnover, recruitment problems, setting and venue concerns, availability of potential participants and participant characteristics were also identified as evaluation roadblocks. Each issue and corresponding salvage strategy is presented.Summary The experiences of conducting such a novel trial as the preliminary Melbourne Diabetes Prevention Study have been invaluable. The lessons learnt and knowledge gained will inform the future execution of this trial in the coming years. We anticipate that these results will also be beneficial to other researchers conducting similar trials in the public health field. We recommend that researchers openly share their experiences, barriers and challenges when conducting randomised controlled trials and implementation research. We encourage them to describe the factors that may have inhibited or enhanced the desired outcomes so that the academic community can learn and expand the research foundation of implementation salvage.<br /

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

publishedVersio

Microbial systematics and taxonomy: relevance for a microbial commons

The issues of microbial taxonomy and potential interactions with a microbial commons are discussed, with emphasis on three components: characterization; classification; and nomenclature. The current state of technology and the spectrum of methods that are used for phenotypic and genotypic characterization of prokaryotes, classification at different taxonomic levels and points of prokaryote nomenclature are reviewed. While all taxonomic ranks comprise a cohesive systematic framework for microorganisms, the prokaryotic genus and species provide the "working unit" of taxonomy. Since 2004, the number of validly published genera and species has increased by approximately 50%. Extensive development of technology will continue to enable ever higher resolution characterization and more refined classification of microorganisms. Characterization and classification at the species level may be most relevant for bacterial taxonomy, although reproducible differentiation at the strain level will probably prove to be more relevant for a microbial commons. A dynamic microbial taxonomy, albeit with well-founded and stable guidelines for defining microorganisms, provides an efficient organizational system for dealing with the enormous spectrum of microbial diversity. (C) 2010 Elsevier Masson SAS. All rights reserved.Antimicrobial treatment and prevention of infection