| 1. |
- Gomila, Margarita, et al.
(författare)
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Genotypic and phenotypic applications for the differentiation and species-level identification of achromobacter for clinical diagnoses.
- 2014
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 9:12
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Tidskriftsartikel (refereegranskat)abstract
- The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected 'house-keeping' genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.
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| 2. |
- Grankvist, Anna, et al.
(författare)
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Multilocus sequence analysis of clinical "candidatus neoehrlichia mikurensis" strains from Europe
- 2015
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 53:10, s. 3126-3132
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Tidskriftsartikel (refereegranskat)abstract
- Copyright © 2015, American Society for Microbiology. All Rights Reserved. Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n9), the Czech Republic (n2), and Germany (n1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.
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| 3. |
- Hedberg, Maria E, et al.
(författare)
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Lachnoanaerobaculum gen. nov., a new genus in the Lachnospiraceae: characterization of Lachnoanaerobaculum umeaense gen. nov., sp. nov., isolated from the human small intestine, and Lachnoanaerobaculum orale sp. nov., isolated from saliva, and reclassification of Eubacterium saburreum (Prevot 1966) Holdeman and Moore 1970 as Lachnoanaerobaculum saburreum comb. nov.
- 2012
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5034 .- 1466-5026. ; 62:Pt 11, s. 2685-90
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Tidskriftsartikel (refereegranskat)abstract
- Two novel obligately anaerobic, Gram-stain-positive, saccharolytic and non-proteolytic spore-forming bacilli (strains CD3 : 22(T) and N1(T)) are described. Strain CD3 : 22(T) was isolated from a biopsy of the small intestine of a child with coeliac disease, and strain N1(T) from the saliva of a healthy young man. The cells of both strains were observed to be filamentous, approximately 5 to >20 µm long, some of them curving and with swellings. The novel organisms produced H(2)S, NH(3), butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98 % sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum and four other Lachnospiraceae bacterium-/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium species are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089(T) confirmed that the bacterium is indeed able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, strains CD3 : 22(T) and N1(T) represent novel species of a new and distinct genus, named Lachnoanaerobaculum gen. nov., in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3 : 22(T) ( = CCUG 58757(T) = DSM 23576(T)) is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1(T) ( = CCUG 60305(T) = DSM 24553(T)) is the type strain of Lachnoanaerobaculum orale sp. nov. Moreover, Eubacterium saburreum is reclassified as Lachnoanaerobaculum saburreum comb. nov. (type strain CCUG 28089(T) = ATCC 33271(T) = CIP 105341(T) = DSM 3986(T) = JCM 11021(T) = VPI 11763(T)).
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| 4. |
- Hedberg, Maria E., et al.
(författare)
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Prevotella jejuni sp. nov., isolated from the small intestine of a child with celiac disease.
- 2013
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5034 .- 1466-5026. ; 63:11, s. 4218-4223
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Tidskriftsartikel (refereegranskat)abstract
- Five obligately anaerobic, Gram-negative, saccharolytic and proteolytic, non-spore-forming bacilli (CD3:27, CD3:28T, CD3:33, CD3:32 and CD3:34) are described. All five strains were isolated from the small intestine of a female child with celiac disease. The cells of the five strains were observed to be short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis, based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3:27, CD3:28T and CD3:33 on one hand, between CD3:32 and P. histicola CCUG 55407T and between CD3:34 and P. melaninogenica CCUG 4944BT on the other. The strains CD3:27, CD3:28T and CD3:33 were clearly different from any other species within the genus Prevotella and most closely related to but distinct from P. melaninogenica. Based on 16S rRNA gene, RNA polymerase β-subunit gene and 60-kDa chaperonin protein subunit gene sequencing, phenotypic, chemical and biochemical properties strains CD3:27, CD3:28T and CD3:33 have been determined to represent a novel species within the genus Prevotella, named Prevotella jejuni sp. nov. Strain CD3:28T (CCUG 60371T = DSM 26989T) is the type strain of the proposed new species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 °C.
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| 5. |
- Isaksson, Jenny, et al.
(författare)
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Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems
- 2015
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Ingår i: Diagnostic Microbiology and Infectious Disease. - : Elsevier BV. - 0732-8893 .- 1879-0070. ; 81:4, s. 240-245
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMerieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp. (C) 2015 Elsevier Inc. All rights reserved.
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| 6. |
- Jaén-Luchoro, Daniel, 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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| 7. |
- Johnning, Anna, 1985, et al.
(författare)
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Acquired genetic mechanisms of a multiresistant bacterium isolated from a treatment plant receiving wastewater from antibiotic production.
- 2013
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Ingår i: Applied and environmental microbiology. - 1098-5336 .- 0099-2240. ; 79:23, s. 7256-63
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Tidskriftsartikel (refereegranskat)abstract
- The external environment, particularly wastewater treatment plants (WWTPs), where environmental bacteria meet human commensals and pathogens in large numbers, has been highlighted as a potential breeding ground for antibiotic resistance. We have isolated the extensively drug-resistant Ochrobactrum intermedium CCUG 57381 from an Indian WWTP receiving industrial wastewater from pharmaceutical production contaminated with high levels of quinolones. Antibiotic susceptibility testing against 47 antibiotics showed that the strain was 4 to >500 times more resistant to sulfonamides, quinolones, tetracyclines, macrolides, and the aminoglycoside streptomycin than the type strain O. intermedium LMG 3301(T). Whole-genome sequencing identified mutations in the Indian strain causing amino acid substitutions in the target enzymes of quinolones. We also characterized three acquired regions containing resistance genes to sulfonamides (sul1), tetracyclines [tet(G) and tetR], and chloramphenicol/florfenicol (floR). Furthermore, the Indian strain harbored acquired mechanisms for horizontal gene transfer, including a type I mating pair-forming system (MPFI), a MOBP relaxase, and insertion sequence transposons. Our results highlight that WWTPs serving antibiotic manufacturing may provide nearly ideal conditions for the recruitment of resistance genes into human commensal and pathogenic bacteria.
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| 8. |
- Karlsson, R., et al.
(författare)
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Proteotyping: Proteomic characterization, classification and identification of microorganisms - A prospectus
- 2015
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Ingår i: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020. ; 38:4, s. 246-257
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Forskningsöversikt (refereegranskat)abstract
- Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels. (C) 2015 Elsevier GmbH. All rights reserved.
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| 9. |
- Karlsson, Roger, et al.
(författare)
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Proteotyping: Tandem Mass Spectrometry Shotgun Proteomic Characterization and Typing of Pathogenic Microorganisms
- 2016
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Ingår i: MALDI‐TOF and Tandem MS for Clinical Microbiology. - Chichester, UK : John Wiley & Sons, Ltd. - 9781118960226 ; , s. 419-450
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Proteotyping provides the means to identify and quantify the actual expression patterns of proteins and their associated pathways, which provides a more accurate picture of infectious agents and their pathogenic potential. Proteotyping, as an analytical method, is intimately correlated with genotypic or genomic data and offers an approach for a holistic characterization of microorganisms. Bioinformatics is vital for the analysis of the data generated by shotgun proteomics. This chapter describes the complete bioinformatics workflow necessary for proteotyping. Mass spectrometry (MS)-based shotgun proteomics analyses offer more detailed and comprehensive analyses of microorganisms. Two approaches may be applied: the so-called top-down and bottom-up proteomics. A major driver for the development and use of tandem MS and proteotyping in clinical settings will be the rapidly growing databases of whole genome reference sequences, which will refine microbial phylogeny and provide a foundation for proteomics-based identification.
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| 10. |
- Karlsson, Roger, 1975, et al.
(författare)
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Strain-level typing and identification of bacteria using mass spectrometry-based proteomics.
- 2012
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 11:5, s. 2710-20
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Tidskriftsartikel (refereegranskat)abstract
- Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC-MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for Helicobacter pylori .
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