| 1. |
- Janzon, Anders, 1978, et al.
(författare)
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Exploring the microbial resistome in river sediments exposed to extraordinary high levels of antibiotics
- 2010
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Ingår i: 35th FEBS Congress: Molecules of Life.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The rapid development and propagation of antibiotic resistance in pathogenic and opportunistic bacteria is a major threat to public health worldwide. The phenomenon has been widely studied in the clinical setting, but comparatively little is known about the prevalence and diversity of antibiotic resistance in communities of environmental bacteria, often referred to as the environmental resistome. As the external environment may function as a reservoir of resistance genes to human pathogens, we are interested in how environmental bacteria are affected by antibiotic pollution. We have previously isolated microbial DNA from river sediments taken up- and downstream from a water treatment plant that processes waste water from several pharmaceutical plants producing antibiotics. In a previous study, we used deep sequencing to identify unprecedented frequencies of known resistance genes to several classes of antibiotics in these samples. In this study, we aim to functionally characterize the resistome in a more open and exploratory way by screening genomic DNA libraries transformed into sensitive hosts. To generate the libraries, several experimental strategies were explored, including mechanical shearing and enzymatic digestion of the isolated DNA followed by blunt- or sticky end cloning into different plasmids, subsequently transformed into sensitive E. coli. Pros and cons of the different strategies will be discussed along with preliminary results of the screening against selected antibiotics.
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| 2. |
- Jaén-Luchoro, Daniel, 1987, et al.
(författare)
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Corynebacterium sanguinis sp. nov., a clinical and environmental associated corynebacterium.
- 2020
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Ingår i: Systematic and applied microbiology. - : Elsevier BV. - 1618-0984 .- 0723-2020. ; 43:1
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Tidskriftsartikel (refereegranskat)abstract
- Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).
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| 3. |
- Méndez, Valentina, et al.
(författare)
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Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants.
- 2020
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 8:11
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Tidskriftsartikel (refereegranskat)abstract
- The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C.michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)-that grouped Chilean strains in three distinct clusters-and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified.
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| 4. |
- Karlsson, Roger, 1975, et al.
(författare)
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Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics
- 2018
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- A range of methodologies may be used for analyzing bacteria, depending on the purpose and the level of resolution needed. The capability for recognition of species distinctions within the complex spectrum of bacterial diversity is necessary for progress in microbiological research. In clinical settings, accurate, rapid and cost-effective methods are essential for early and efficient treatment of infections. Characterization and identification of microorganisms, using, bottom-up proteomics, or "proteotyping", relies on recognition of species-unique or associated peptides, by tandem mass spectrometry analyses, dependent upon an accurate and comprehensive foundation of genome sequence data, allowing for differentiation of species, at amino acid-level resolution. In this study, the high resolution and accuracy of MS/MS-based proteotyping was demonstrated, through analyses of the three phylogenetically and taxonomically most closely-related species of the Mitis Group of the genus Streptococcus: i.e., the pathogenic species, Streptococcus pneumoniae (pneumococcus), and the commensal species, Streptococcus pseudopneumoniae and Streptococcus mitis. To achieve high accuracy, a genome sequence database used for matching peptides was created and carefully curated. Here, MS-based, bottom-up proteotyping was observed and confirmed to attain the level of resolution necessary for differentiating and identifying the most-closely related bacterial species, as demonstrated by analyses of species of the Streptococcus Mitis Group, even when S. pneumoniae were mixed with S. pseudopneumoniae and S. mitis, by matching and identifying more than 200 unique peptides for each species.
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| 5. |
- Gomila, Margarita, et al.
(författare)
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Kinneretia asaccharophila gen. nov., sp. nov., isolated from a freshwater lake, a member of the Rubrivivax branch of the family Comamonadaceae.
- 2010
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 60:4, s. 809-814
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Tidskriftsartikel (refereegranskat)abstract
- A strictly aerobic, Gram-negative bacterium, strain KIN192(T), isolated from fresh water from Lake Kinneret, Israel, was examined using a polyphasic approach to characterize and clarify its phylogenetic and taxonomic position. Sequences of the 16S rRNA and gyrB genes and ITS1 revealed close relationships to species of the genera Pelomonas, Mitsuaria and Roseateles, in the Rubrivivax branch of the family Comamonadaceae of the Betaproteobacteria. Physiological and biochemical tests, cellular fatty acid analysis and DNA-DNA hybridizations indicated that this strain should be assigned to a new genus and species in the Rubrivivax phylogenetic branch, for which the name Kinneretia asaccharophila gen. nov., sp. nov., is proposed. The type strain of Kinneretia asaccharophila is strain KIN192(T) (=CCUG 53117(T) =CECT 7319(T)).
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| 6. |
- Salvà-Serra, Francisco, 1989, et al.
(författare)
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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
- 2023
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- 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-beta-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 beta-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic beta-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic beta-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.
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| 7. |
- Stevenson, Bradley S, et al.
(författare)
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Hoeflea anabaenae sp. nov., an epiphytic symbiont that attaches to the heterocysts of a strain of Anabaena.
- 2011
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5034 .- 1466-5026. ; 61:10, s. 2439-2444
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Tidskriftsartikel (refereegranskat)abstract
- The heterotrophic, epiphytic symbiotic bacterial strain WH2K(T) was previously isolated from a two-member culture in which it was attached to the heterocysts of a strain of Anabaena (SSM-00). Analysis of its 16S rRNA genes demonstrated that the symbiont was most closely related to the type strain of Hoeflea marina (96.9% similarity), which belongs to the family Phyllobacteriaceae within the order Rhizobiales of the class Alphaproteobacteria. A polyphasic taxonomic study was performed on strain WH2K(T) that consisted of irregular rods (2-5μm long, 0.2μm wide) that appeared to be narrower at one polar end. Optimal growth was obtained in complex media with 15 g l(-1) sea salts, at 18-34 °C (30 °C optimum) and at pH values in the range of 6.0-8.0 (6.5 optimum). Unknown growth requirements were provided by small amounts of yeast extract but not standard vitamin and trace metal solutions. Of the substrates tested, WH2K(T) was only able to utilize acetate, pyruvate, malate, and fumarate. Growth was only observed under aerobic and microaerobic conditions and nitrate was not reduced. No photosynthetic pigments were detected under any of the growth conditions tested. The predominant fatty acids were present in a summed feature that comprises C (18:1) ω7c, / 9t /12t or any combination of these (64.0%) and an unidentified fatty acid of estimated chain length (ECL of C(17.6) (13.5%), The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphoglycolipid, unknown lipids and an unidentified aminolipid. The only respiratory ubiquinone detected was Q-10. The DNA %G+C content of the strain is 58.1%. The organism can form a site-specific attached symbiotic relationship with a species of Anabaena. Based on phylogenetic and phenotypic evidence it is proposed that strain WH2K(T) be classified as a fourth species of the genus Hoeflea, for which the name Hoeflea anabaenae sp. nov. is proposed. The type strain of Hoeflea anabaenae sp. nov. is WH2K(T) (=CCUG 56626(T) =NRRL B-59520(T)).
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| 8. |
- Vaz-Moreira, Ivone, et al.
(författare)
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Shinella fusca sp. nov., isolated from domestic waste compost
- 2010
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 60:1, s. 144-148
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Tidskriftsartikel (refereegranskat)abstract
- A bacterium, designated strain DC-196(T), isolated from kitchen refuse compost was analysed by using a polyphasic approach. Strain DC-196(T) was characterized as a Gram-negative short rod that was catalase- and oxidase-positive, and able to grow at 10-40 degrees C, pH 6-9 and in NaCl concentrations as high as 3 %. Chemotaxonomically, C(18 : 1) was observed to be the predominant cellular fatty acid and ubiquinone 10 (Q10) was the predominant respiratory quinone. The G+C content of the genomic DNA was determined to be 66 mol%. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, strain DC-196(T) was assigned to the genus Shinella, although with distinctive features. At the time of writing, 16S rRNA gene sequence similarities of 97.6-96.8 % and the low DNA-DNA hybridization values of 38.2-32.2 % with the type strains of the three recognized Shinella species confirmed that strain DC-196(T) represents a novel species of the genus, for which the name Shinella fusca sp. nov. is proposed (type strain DC-196(T)=CCUG 55808(T)=LMG 24714(T)).
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| 9. |
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| 10. |
- Gonzales-Siles, Lucia, et al.
(författare)
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Mass Spectrometry Proteotyping for detection, identification characterization and diagnostics of infectious bacteria in clinical respiratory-tract samples
- 2016
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Ingår i: 11th International Meeting on Microbial Epidemiological Markers (IMMEM XI) 9 - 12 March 2016, Estoril, Portugal.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background. Lower respiratory tract infection (LRTI) is the leading cause of childhood deaths in most developing countries and the world (?) and are the most common causes of hospital and out-patient visits within the EU, comprising 1 of 3 admissions annually. In general, the over-prescription and use of broad-spectrum antibiotics are common practices that lead to the evolution and development of resistance in infectious bacteria and will lead to loss of time and resources in patient handling and adverse patient outcomes. Conventional approaches have depended upon cultivation of bacteria with subsequent testing for antibiotic sensitivity. Therefore, reliable and time-effective microbiological diagnostics are essential for more effective treatment of respiratory infections. In this project, we apply state-of-the-art proteomics techniques for identifications of pathogens and antibiotic resistance from clinical samples, without prior cultivation. Material and methods. Nasopharyngeal swab samples were collected, in commercial Amies medium supplemented with 5x STGG, as a part of the EU-TAILORED-Treatment project (www.tailored-treatment.eu/). Samples were stored at -20°C until analyses. Different protocols for removal of human cells and mucus were tested, including non-ionic detergents, i.e., Igepal, Saponin, Urea-Chaps, as well as cytolysis. Samples were concentrated and analyzed by ‘proteotyping’ (1), using a Lipid-based Protein Immobilization (LPITM) technology (WO2006068619), in which intact bacterial cells or cell fractions are bound to a surface. Peptides were generated, using enzymatic digestion, and then separated and analyzed, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass spectra profiles were compared to a database of reference peptide sequences, consisting of all complete genomes of the NCBI Reference Sequence (RefSeq) Database. Results were confirmed by standard microbiology, including cultivation of bacteria in selective media, MALDI-TOF MS analyses and qPCR. Results. Proteotyping applied to clinical samples demonstrated that the number of viable bacteria and detected proteins determined were ten-times higher when nasal swabs were stored in Amies media supplemented with STGG 5X media compared to Amies media without STGG, after 1 and 2 months of storage at -70C. Among the different protocols tested to remove human biomaterial, all treatments proved effective to varying degrees, although the Igepal treatment was able to retain the highest number of discriminatory peptides. Using proteotyping, we were able to identify the pathogenic bacteria directly within clinical samples (nasopharyngeal and nasal swabs) that had been identified to be positive for respiratory infectious bacteria by standard methodologies at clinical bacteriology laboratories at Sahlgrenska University Hospital (Sweden) or Universitair Medisch Centrum Utrecht (Netherlands). Conclusions. Proteotyping of infectious bacteria, using tandem LC-MS/MS enabled the differentiation and identification of infectious bacteria in clinical samples from LRTIs. It has high levels of resolution and highly reproducible detection of protein biomarkers. Proteotyping identified biomarkers for species- and sub-species-level strain discrimination and antibiotic resistance, all from single MS analyses. 1) Karlsson et al., 2015. Syst. Appl. Microbiol. 38 :246-257.
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