Dynamics of Resistance Plasmids in Extended-Spectrum-β-Lactamase-Producing during Postinfection Colonization.

Extended-spectrum β-lactamase-producing (EPE) are a major cause of bloodstream infections, and the colonization rate of EPE in the gut microbiota of individuals lacking prior hospitalization or comorbidities is increasing. In this study, we performed an in-depth investigation of the temporal dynamics of EPE and their plasmids during one year by collecting fecal samples from three patients initially seeking medical care for urinary tract infections. In two of the patients, the same strain that caused the urinary tract infection (UTI) was found at all consecutive samplings from the gut microbiota, and no other EPEs were detected, while in the third patient the UTI strain was only found in the initial UTI sample.

Ancient permafrost staphylococci carry antibiotic resistance genes.

: Permafrost preserves a variety of viable ancient microorganisms. Some of them can be cultivated after being kept at subzero temperatures for thousands or even millions of years. : To cultivate bacterial strains from permafrost.

Network Experiences from a Cross-Sector Biosafety Level-3 Laboratory Collaboration: A Swedish Forum for Biopreparedness Diagnostics.

The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories.

A suggested new bacteriophage genus, "Kp34likevirus", within the Autographivirinae subfamily of Podoviridae.

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418).

A multicentre hospital outbreak in Sweden caused by introduction of a vanB2 transposon into a stably maintained pRUM-plasmid in an Enterococcus faecium ST192 clone.

The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n = 18) and vancomycin-susceptible E. faecium (VSEfm, n = 2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n = 3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer.

Draft Genome Sequence of Pseudomonas aeruginosa SG17M, an Environmental Isolate Belonging to Clone C, Prevalent in Patients and Aquatic Habitats.

Pseudomonas aeruginosa SG17M is an environmental isolate recovered from river water in the city of Mulheim, Germany. SG17M belongs to clone C, which is distributed worldwide. This is the first clone C strain whose genome sequence has been determined.

The need for high-quality whole-genome sequence databases in microbial forensics.

Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses.

Plasmidome-analysis of ESBL-producing escherichia coli using conventional typing and high-throughput sequencing.

Infections caused by Extended spectrum β-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used β-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements.

Adaptive mutations and replacements of virulence traits in the Escherichia coli O104:H4 outbreak population.

The sequencing of highly virulent Escherichia coli O104:H4 strains isolated during the outbreak of bloody diarrhea and hemolytic uremic syndrome in Europe in 2011 revealed a genome that contained a Shiga toxin encoding prophage and a plasmid encoding enteroaggregative fimbriae. Here, we present the draft genome sequence of a strain isolated in Sweden from a patient who had travelled to Tunisia in 2010 (E112/10) and was found to differ from the outbreak strains by only 38 SNPs in non-repetitive regions, 16 of which were mapped to the branch to the outbreak strain. We identified putatively adaptive mutations in genes for transporters, outer surface proteins and enzymes involved in the metabolism of carbohydrates.

Genome sequencing reveals a phage in Helicobacter pylori.

Unlabelled: Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled.

Titration-free 454 sequencing using Y adapters.

We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe-based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment.

Genomic stability over 9 years of an isoniazid resistant Mycobacterium tuberculosis outbreak strain in Sweden.

In molecular epidemiological studies of drug resistant Mycobacterium tuberculosis (TB) in Sweden a large outbreak of an isoniazid resistant strain was identified, involving 115 patients, mainly from the Horn of Africa. During the outbreak period, the genomic pattern of the outbreak strain has stayed virtually unchanged with regard to drug resistance, IS6110 restriction fragment length polymorphism and spoligotyping patterns. Here we present the complete genome sequence analyses of the index isolate and two isolates sampled nine years after the index case as well as experimental data on the virulence of this outbreak strain.

Titration-free massively parallel pyrosequencing using trace amounts of starting material.

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.

Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.

Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins.

The Escherichia coli CsrB and CsrC small RNAs are strongly induced during growth in nutrient-poor medium.

The carbon storage regulatory (Csr) system is a complex network controlling various phenotypes in many eubacteria. So far, the external conditions by which the system is regulated are poorly understood. Here we show that the expression of the two noncoding small RNAs CsrB and CsrC in Escherichia coli is strongly increased in cultures grown in minimal medium.

Regulation of c-di-GMP metabolism in biofilms.

Cyclic (5 to 3 )-diguanosine monophosphate (c-di-GMP) is a small molecule that regulates the transition between the sessile and motile lifestyle, an integrative part of biofilm formation and other multicellular behavior, in many bacteria. The recognition of c-di-GMP as a novel secondary messenger soon raised the question about the specificity of the signaling system, as individual bacterial genomes frequently encode numerous c-di-GMP metabolizing proteins. Recent work has demonstrated that several global regulators concertedly modify the expression of selected panels of c-di-GMP metabolizing proteins, which act on targets with physiological functions.

Sortase-mediated assembly and surface topology of adhesive pneumococcal pili.

The rlrA genetic islet encodes an extracellular pilus in the Gram-positive pathogen Streptococcus pneumoniae. Of the three genes for structural subunits, rrgB encodes the major pilin, while rrgA and rrgC encode ancillary pilin subunits decorating the pilus shaft and tip. Deletion of all three pilus-associated sortase genes, srtB, srtC and srtD, completely prevents pilus biogenesis.

The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteins.

The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains.

An Escherichia coli asr mutant has decreased fitness during colonization in a mouse model.

The Escherichia coli asr gene, like its homologues in other enterobacteria, is strongly induced by low external pH. The E. coli asr mutant shows weakened ability to adapt to acidic pH.

Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy.

Background: Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy.

Results: AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures.

Identification of YhdA as a regulator of the Escherichia coli carbon storage regulation system.

The Escherichia coli BarA-UvrY two-component system, which controls adaptation via the CsrB and CsrC sRNAs, is induced at the entry of the stationary phase by an unknown stimulus. Using a csrB-lacZ fusion, we demonstrated that the factors RelA, SpoT and LuxS, previously suggested to act on orthologues of this system, have no role in BarA-UvrY induction. However, using a transposon screen, we identified the hypothetical protein YhdA as a new regulator of CsrB and CsrC expression.

pH-dependent activation of the BarA-UvrY two-component system in Escherichia coli.

The barA and uvrY genes of Escherichia coli encode a two-component sensor kinase and a response regulator, respectively. Although this system plays a major role in the regulation of central carbon metabolism, motility, and biofilm formation by controlling the expression of the CsrB and CsrC noncoding RNAs, the environmental conditions and the physiological signal(s) to which it responds remain obscure. In this study, we explored the effect of external pH on the activity of BarA/UvrY.

Of two cytosolic aconitases expressed in Drosophila, only one functions as an iron-regulatory protein.

In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs.

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract.

Background: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model.

Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype.

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors.