Repeated introduction and spread of the MRSA clone t304/ST6 in northern Europe.

OBJECTIVES: During the last decades several methicillin-resistant Staphylococcus aureus (MRSA) clones with the capability of global spread have emerged in the community. Here, we have investigated a large collection of clinical isolates belonging to MRSA clone t304/ST6, which has emerged in many European countries over the last years, in order to retrace its phylogeny and its spread. METHODS: We characterized 466 ST6 isolates from Denmark (n = 354), France (n = 10), Norway (n = 24), Sweden (n = 27) and the UK (n = 51). All had spa-type t304 (n = 454) or t304-related spa-types (n = 12) and whole genome sequencing (WGS) was carried out on Illumina Miseq or Hiseq with 100-300 bp reads. cgMLST was performed using Ridom SeqSphere. RESULTS: A minimum spanning tree (MST) of all 466 isolates showed one large cluster including 182 isolates collected only from Denmark and related to a long-term neonatal outbreak in Copenhagen. This cluster contrasted with numerous small clusters, including the remaining Danish isolates and isolates from the other countries that interspersed throughout the tree. Most isolates were Panton-Valentine leukocidin (PVL) negative (95%) and harboured SCCmec IVa. One genome was closed using Oxford Nanopore technology and Illumina MiSeq. It contained a plasmid of 19.769 bp including the blaZ gene. A similar plasmid was found in 78% of all isolates. DISCUSSION: t304/ST6 is a successful emerging clone and the fact that isolates from five countries are interspersed throughout the MST indicates a common origin. This clone is commonly described in the Middle East and its emergence in Europe coincides with influx of refugees from the Syrian Civil War.

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries.

OBJECTIVES: Sample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination. RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.

A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus.

BACKGROUND: The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. OBJECTIVE: To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. STUDY DESIGN: In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. RESULTS: The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. CONCLUSIONS: The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

Effects of sampling standardization on estimates of Phanerozoic marine diversification.

Global diversity curves reflect more than just the number of taxa that have existed through time: they also mirror variation in the nature of the fossil record and the way the record is reported. These sampling effects are best quantified by assembling and analyzing large numbers of locality-specific biotic inventories. Here, we introduce a new database of this kind for the Phanerozoic fossil record of marine invertebrates. We apply four substantially distinct analytical methods that estimate taxonomic diversity by quantifying and correcting for variation through time in the number and nature of inventories. Variation introduced by the use of two dramatically different counting protocols also is explored. We present sampling-standardized diversity estimates for two long intervals that sum to 300 Myr (Middle Ordovician-Carboniferous; Late Jurassic-Paleogene). Our new curves differ considerably from traditional, synoptic curves. For example, some of them imply unexpectedly low late Cretaceous and early Tertiary diversity levels. However, such factors as the current emphasis in the database on North America and Europe still obscure our view of the global history of marine biodiversity. These limitations will be addressed as the database and methods are refined.

Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria.

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.

Fermentative bacteria from estuarine mud: phylogenetic position of Acidaminobacter hydrogenoformans and description of a new type of gram-negative, propionigenic bacterium as Propionibacter pelophilus gen. nov., sp. nov.

The phylogenetic positions of two strains of fermentative bacteria that had been isolated from the highest positive tubes inoculated with serial dilutions of estuarine mud in agar media with either glutamate or aspartate as substrate were determined by comparative sequence analysis of their 16S rRNA genes. The strain isolated with glutamate (glu 65) utilized several substrates, including a number of amino acids but no sugars. The degradation of certain substrates was enhanced by or dependent upon co-cultivation with a hydrogen-utilizing partner. In earlier work this strain was assigned to the new genus and species Acidaminobacter hydrogenoformans. On the basis of its 16S rRNA gene sequence Acidaminobacter hydrogenoformans has now been identified as a member of cluster XI of the Clostridium subphylum with Clostridium halophilum as its closest relative. The aspartate-fermenting strain asp 66T was a Gram-negative, rather aerotolerant anaerobe which utilized a wide range of substrates in a propionic fermentation and had the ability to fix molecular nitrogen. Strain asp 66T was shown to be a new member of the beta-subclass of the Proteobacteria with Azoarcus sp. strain 6a3 and Rhodocyclus tenuis as its closest relatives. It is described as Propionibacter pelophilus gen. nov., sp. nov., with the type strain asp 66T (= DSM 12018T).

Tetrahydrofolate serves as a methyl acceptor in the demethylation of dimethylsulfoniopropionate in cell extracts of sulfate-reducing bacteria.

Tetrahydrofolate was shown to function as a methyl acceptor in the anaerobic demethylation of dimethylsulfoniopropionate to methylthiopropionate in cell extracts of the sulfate-reducing bacterium strain WN. Dimethylsulfoniopropionate-dependent activities were 0.56 micromol methyltetrahydrofolate min-1 (mg protein)-1 and were higher than required to explain the growth rate of strain WN on dimethylsulfoniopropionate. The reaction did not require ATP or reductive activation by titanium(III)-nitrilotriacetic acid. Preincubation of the extract under air significantly decreased the activity (35% loss in 3 h). Three other dimethylsulfoniopropionate-demethylating sulfate reducers, Desulfobacterium niacini, Desulfobacterium vacuolatum, and Desulfobacterium strain PM4, had dimethylsulfoniopropionate:tetrahydrofolate methyltransferase activities of 0.16, 0.05, and 0.24 micromol min-1 (mg protein)-1, respectively. No methyltransferase activity to tetrahydrofolate was found with betaine as a substrate, not even in extracts of betaine-grown cells of these sulfate reducers. Dimethylsulfoniopropionate demethylation in cell extracts of strain WN was completely inhibited by 0.5 mM propyl iodide; in the light, the inhibition was far less strong, indicating involvement of a corrinoid-dependent methyltransferase.

Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate).

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D. gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate).

Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria.

The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate. Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp. strain PM4. The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains. Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum. From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated. Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids. Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway. Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica. Strain PM4 was shown to group with Desulfobacterium niacini. The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM. A similar value was found for D. niacini PM4.

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Methanogenic conversion of 3-s-methylmercaptopropionate to 3-mercaptopropionate.

Anaerobic metabolism of dimethylsulfoniopropionate, an osmolyte of marine algae, in anoxic intertidal sediments involves either cleavage to dimethylsulfide or demethylation to 3-S-methylmercaptopropionate (MMPA) and subsequently to 3-mercaptopropionate. The methanogenic archaea Methanosarcina sp. strain MTP4 (DSM 6636), Methanosarcina acetivorans DSM 2834, and Methanosarcina (Methanolobus) siciliae DSM 3028 were found to use MMPA as a growth substrate and to convert it stoichiometrically to 3-mercaptopropionate. Approximately 0.75 mol of methane was formed per mol of MMPA degraded; methanethiol was not detected as an intermediate. Eight other methanogenic strains did not carry out this conversion. We also studied the conversion of MMPA in anoxic marine sediment slurries. Addition of MMPA (500 (mu)M) resulted in the production of methanethiol which was subsequently converted to methane (417 (mu)M). In the presence of the antibiotics ampicillin, vancomycin, and kanamycin (20 (mu)g/ml each), 275 (mu)M methane was formed from 380 (mu)M MMPA; no methanethiol was formed during these incubations. Only methanethiol was formed from MMPA when 2-bromoethanesulfonate (25 mM) was added to a sediment suspension. These results indicate that in natural environments MMPA could be directly or indirectly a substrate for methanogenic archaea.

Metabolism of sulfate-reducing prokaryotes.

Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 degrees C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO2. The most detailed knowledge is available on the metabolism of Desulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.

Involvement of an Intracellular Oligogalacturonate Hydrolase in Metabolism of Pectin by Clostridium thermosaccharolyticum.

The enzymes pectin methylesterase and polygalacturonate hydrolase, which are responsible for the initial steps of pectin degradation by Clostridium thermosaccharolyticum, were shown to be induced on the polymeric substrates pectin and pectate, as well as on oligogalacturonates, and to be repressed in the presence of glucose. The digalacturonate and trigalacturonate produced by the extracellular pectin methylesterase-polygalacturonate hydrolase complex were transported across the cytoplasmic membrane and hydrolyzed by an inducible oligogalacturonate hydrolase to galacturonate. The oligogalacturonate hydrolase was separated from the polygalacturonate hydrolase and characterized. Its temperature optimum was 65 degrees C, and its pH optimum was 6. The native molecular size was 90 kDa, and the enzyme was stable for more than 1 h at 65 degrees C. The maximum reaction rate on oligomers decreased with the increasing degree of polymerization. Galacturonate was released by hydrolysis from the nonreducing end of the oligomer. The amounts of pectinolytic enzymes produced were all strictly correlated to the amount of biomass formed. Galacturonate was metabolized via a modified Entner-Doudoroff route.

Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.

Betaine fermentation and oxidation by marine desulfuromonas strains.

Two bacterial strains were dominant in anaerobic enrichment cultures with betaine (N,N,N-trimethylglycine) as a substrate and intertidal mud as an inoculum. One was a coccoid bacterium which was a trimethylamine (TMA)-fermenting methanogen similar to Methanococcoides methylutens. The other strain, a rod-shaped, gram-negative, motile bacterium, fermented betaine. On the basis of its ability to oxidize acetate and ethanol to CO(2) with sulfur as an electron acceptor, its inability to reduce sulfate and sulfite, its morphology, the presence of c-type cytochromes, and other characteristics, the isolated strain PM1 was identified as Desulfuromonas acetoxidans. Although only malate and fumarate were known as substrates for fermentative growth of this species, the type strain (DSM 684) also fermented betaine. Strain PM1 grew with a doubling time of 9.5 h at 30 degrees C on betaine and produced approximately 1 mol of TMA per mol of betaine, 0.75 mol of acetate, and presumably CO(2) as fermentation products but only in the presence of selenite (100 nM). In this fermentation, betaine is probably reductively cleaved to TMA and acetate, and part of the acetate is then oxidized to CO(2) to provide the reducing equivalents for the initial cleavage reaction. In the presence of sulfur, betaine was converted to TMA and presumably CO(2) with the formation of sulfide; then, only traces of acetate were produced.

A tsunami deposit at the cretaceous-tertiary boundary in Texas.

At sites near the Brazos River, Texas, an iridium anomaly and the paleontologic Cretaceous-Tertiary boundary directly overlie a sandstone bed in which coarse-grained sandstone with large clasts of mudstone and reworked carbonate nodules grades upward to wave ripple-laminated, very fine grained sandstone. This bed is the only sandstone bed in a sequence of uppermost Cretaceous to lowermost Paleocene mudstone that records about 1 million years of quiet water deposition in midshelf to outer shelf depths. Conditions for depositing such a sandstone layer at these depths are most consistent with the occurrence of a tsunami about 50 to 100 meters high. The most likely source for such a tsunami at the Cretaceous-Tertiary boundary is a bolidewater impact.