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  • 1.
    Becker, Fiona
    et al.
    Novozymes A/S, Bagsværd, Denmark.
    Schnorr, Kirk
    Novozymes A/S, Bagsværd, Denmark.
    Wilting, Reinhard
    Novozymes A/S, Bagsværd, Denmark.
    Tolstrup, Niels
    Exiqon, Vedbaek, Denmark.
    Bendtsen, Jannick Dyrløv
    Center for Biological Sequence Analysis, The Technical University of Denmark, Lyngby.
    Olsen, Peter Bjarke
    Novozymes A/S, Bagsværd, Denmark.
    Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries2004In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 57, no 1, p. 123-133Article in journal (Refereed)
  • 2. Bjerketorp, J.
    et al.
    Ng Tze Chiang, A.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Micro Structural Technology.
    Rosenquist, M.
    Liu, Wen-Tso
    Jansson, J. K.
    Rapid lab-on-a-chip profiling of human gut bacteria2008In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 72, no 1, p. 82-90Article in journal (Refereed)
    Abstract [en]

    The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.

  • 3.
    Bunikis, Ignas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bonde, Mari
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kutschan-Bunikis, Sabrina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi.2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 86, no 2, p. 243-7Article in journal (Refereed)
    Abstract [en]

    Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.

  • 4.
    COUGHLAN, MP
    et al.
    DALGETY UK LTD,CTR RES and TECHNOL,CAMBRIDGE CB1 2JN,ENGLAND; CRANFIELD INST TECHNOL,CTR BIOTECHNOL,DIV BIOELECTR,CRANFIELD MK43 0AL,BEDS,ENGLAND; .
    KIERSTAN, MPJ
    DALGETY UK LTD,CTR RES and TECHNOL,CAMBRIDGE CB1 2JN,ENGLAND; CRANFIELD INST TECHNOL,CTR BIOTECHNOL,DIV BIOELECTR,CRANFIELD MK43 0AL,BEDS,ENGLAND; .
    BORDER, PM
    DALGETY UK LTD,CTR RES and TECHNOL,CAMBRIDGE CB1 2JN,ENGLAND; CRANFIELD INST TECHNOL,CTR BIOTECHNOL,DIV BIOELECTR,CRANFIELD MK43 0AL,BEDS,ENGLAND; .
    TURNER, APF
    Cranfield University, UK.
    ANALYTICAL APPLICATIONS OF IMMOBILIZED PROTEINS AND CELLS1988In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 8, no 02-janArticle, review/survey (Refereed)
    Abstract [en]

    n/a

  • 5.
    Danilov, Roman
    et al.
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Natural Sciences.
    Ekelund, Nils
    Mid Sweden University, Faculty of Science, Technology and Media, Department of Natural Sciences.
    Comparison of usefulness of three types of artificial substrata (glass, wood and plastic) when studying settlement patterns of periphyton in lakes of different trophic status2001In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 45, no 3, p. 167-170Article in journal (Refereed)
    Abstract [en]

    Usefulness of three types of artificial substrata (glass, wood and plastic) was tested when studying settlement patterns of periphyton in lakes of different trophic status. Strictly eu-, meso- and oligotrophic lakes in central Sweden were chosen as objects of the study. Glass slides, glass tubes, pieces of plastic (PVC) and pieces of wood of similar dimensions were placed for 9 weeks in July-August vertically 3 cm above bottom at a total depth of ca. 30 cm. Substrata were located at well-illuminated places without any other submerged objects (like macrophytes and stones), which could potentially affect colonisation patterns by algae. Periphyton communities, which colonised both the glass tubes and the pieces of wood tested, were specific enough to enable a clear classification of the lakes studied in eu-, meso- and oligotrophic. Glass tubes turned out to be the most favourable substratum when investigating settlement patterns of periphyton in this study. Although also colonised by periphytic species, wood did not support the same diversity and abundance of species as glass did. No algae were detected on the plastics studied. The plastics were covered entirely by a slime layer of bacteria. It is discussed if the nature of plastics could have some inhibitory effects on algal growth or the slime layer itself may have prevented settlement of algal spores.

  • 6.
    Dopson, Mark
    et al.
    University of East Anglia, Norwich, UK.
    Baker-Austin, Craigh
    University of East Anglia, Norwich, UK.
    Bond, Philip. L.
    University of East Anglia, Norwich, UK.
    First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships2004In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 58, no 3, p. 297-302Article in journal (Refereed)
    Abstract [en]

    Methods for microbial classification are not always capable of distinguishing between isolates at the species level. We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences.. and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data. In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates. From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities.

  • 7. Eriksson, Ronnie
    et al.
    Jobs, Magnus
    Dalarna University, School of Education, Health and Social Studies, Medical Science.
    Ekstrand, Charlotta
    Ullberg, Måns
    Hermann, Björn
    Landegren, Ulf
    Nilsson, Mats
    Blomberg, Jonas
    Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real-time PCR and specific suspension array readout2009In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 78, no 2, p. 195-202Article in journal (Refereed)
    Abstract [en]

    A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex™ technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex™ detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

  • 8.
    Eriksson, Ronnie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Jobs, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Ekstrand, Charlotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Ullberg, Måns
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout2009In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 78, no 2, p. 195-202Article in journal (Refereed)
    Abstract [en]

    A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

  • 9.
    Fridlund, Jimmy
    et al.
    Kalmar County Hospital, Sweden.
    Woksepp, Hanna
    Kalmar County Hospital, Sweden; Linnaeus University, Sweden.
    Schön, Thomas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Kalmar County Hospital, Sweden; Linnaeus University, Sweden.
    A microbiological method for determining serum levels of broad spectrum beta-lactam antibiotics in critically ill patients2016In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 129, p. 23-27Article in journal (Refereed)
    Abstract [en]

    Background: Recent studies show that suboptimal blood levels of beta-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring beta-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. Methods: The method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n = 88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. Results: The overall accuracy and precision for spiked samples and patient samples was within the pre-set target of +/- 20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r = 0.86, n = 31; MER: r = 0.96, n = 11; PIP: r = 0.88, n = 39) and the agreement around the clinical cut-off for CTX (4.0 mg/l), MER (2.0 mg/l) and PIP (16.0 mg/l) was 90%, 100% and 87%, respectively. Conclusion: The microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory. (C) 2016 Elsevier B.V. All rights reserved.

  • 10.
    Fridlund, Jimmy
    et al.
    Kalmar County Hospital.
    Woksepp, Hanna
    Linnaeus University, Faculty of Health and Life Sciences, Department of Medicine and Optometry. Kalmar County Hospital.
    Schön, Thomas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Medicine and Optometry. Kalmar County Hospital ; Linköping University.
    A microbiological method for determining serum levels of broad spectrum β-lactam antibiotics in critically ill patients2016In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 129, p. 23-27Article in journal (Refereed)
    Abstract [en]

    Background Recent studies show that suboptimal blood levels of β-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring β-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. Methods The method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n = 88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. Results The overall accuracy and precision for spiked samples and patient samples was within the pre-set target of ± 20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r = 0.86, n = 31; MER: r = 0.96, n = 11; PIP: r = 0.88, n = 39) and the agreement around the clinical cut-off for CTX (4.0 mg/l), MER (2.0 mg/l) and PIP (16.0 mg/l) was 90%, 100% and 87%, respectively. Conclusion The microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory.

  • 11.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hamsten, Carl
    KTH, School of Biotechnology (BIO).
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum2010In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 82, no 1, p. 11-18Article in journal (Refereed)
    Abstract [en]

    Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.

  • 12. Gantner, S
    et al.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alonso-Saez, L
    Bertilsson, S
    Novel primers for 16S rRNA-based archaeal community analyses in environmental samples2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, no 1, p. 12-18Article in journal (Refereed)
    Abstract [en]

    Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.

  • 13.
    Gantner, Stephan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Andersson, Anders F.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Alonso-Saez, Laura
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Bertilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Novel primers for 16S rRNA-based archaeal community analyses in environmental samples2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, no 1, p. 12-18Article in journal (Refereed)
    Abstract [en]

    Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 165 rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340E-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.

  • 14.
    Gorokhova, Elena
    et al.
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM). Stockholm University, Faculty of Science, Department of Systems Ecology.
    Mattsson, Lisa
    Stockholm University, Faculty of Science, Department of Systems Ecology.
    Sundström, Annica M.
    Stockholm University, Faculty of Science, Department of Systems Ecology.
    A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy2012In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 89, no 3, p. 216-221Article in journal (Refereed)
    Abstract [en]

    Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by similar to 25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80 degrees C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies.

  • 15. Ihalin, R
    et al.
    Asikainen, Sirkka
    Umeå University, Faculty of Medicine, Odontology, Oral Microbiology.
    16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.2006In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 65, no 3, p. 417-424Article in journal (Refereed)
    Abstract [en]

    Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.

  • 16.
    Isaksson, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Gallo Vaulet, Lucia
    Univ Buenos Aires, Fac Farm & Bioquim, Catedra Microbiol Clin, Junin 956, RA-1113 Buenos Aires, DF, Argentina..
    Christerson, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Ruettger, Anke
    Friedrich Loeffler Inst, Fed Res Inst Anim Hlth, Inst Mol Pathogenesis, D-07743 Jena, Germany..
    Sachse, Konrad
    Friedrich Loeffler Inst, Fed Res Inst Anim Hlth, Inst Mol Pathogenesis, D-07743 Jena, Germany..
    Entrocassi, Carolina
    Univ Buenos Aires, Fac Farm & Bioquim, Catedra Microbiol Clin, Junin 956, RA-1113 Buenos Aires, DF, Argentina..
    Castro, Erica
    Univ San Sebastian, Fac Med, Lientur 1457, Concepcion, Chile..
    Rodriguez Fermepin, Marcelo
    Univ Buenos Aires, Fac Farm & Bioquim, Catedra Microbiol Clin, Junin 956, RA-1113 Buenos Aires, DF, Argentina..
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile2016In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 127, p. 214-218Article in journal (Refereed)
    Abstract [en]

    This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C trachomatis positive specimens were analyzed by ompA sequencing and MIST and of these 76 by MLT array. Obtained MIST sequence types (STs) were compared to sequences in the database http://mIstdb.uu.se. The resolution obtained for MIST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 13 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MIST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.

  • 17.
    Karched, M
    et al.
    Umeå University, Faculty of Medicine, Odontology.
    Paul-Satyaseela, M
    Umeå University, Faculty of Medicine, Odontology.
    Asikainen, Sirkka
    Umeå University, Faculty of Medicine, Odontology, Oral Microbiology.
    A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans2007In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 68, no 1, p. 46-51Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Tenacious adherence and autoaggregation of wild-type Actinobacillus actinomycetemcomitans strains jeopardize reliability of determined cell concentrations, e.g. for studies on bacteria-host interactions. We first compared the efficacy of two methods, an indirect and a direct method, for homogenizing cell suspensions of a wild-type, autoaggregating (SA269) strain and of a non-autoaggregating laboratory variant (ATCC 43718) used as a reference. Since the direct method left visible clumps in SA269 suspension, only the indirect method was further tested. In serial dilutions of the homogenized cell suspensions of strains SA269 and ATCC 43718, the OD(600) values (R(2)=0.99, R(2)=0.99, respectively) and protein concentrations (R(2)=0.93, R(2)=0.95, respectively) correlated significantly (all P<0.002) with the dilution factor. There were no differences (P>0.05) in the bacterial viable counts between the two strains or between suspending solutions, i.e., PBS and water, the cell concentrations demonstrating 1x10(9) cells/ml at OD(600)=1. Repeated microscopic cell counts did not differ (P>0.05) from each other. Large aggregates occurred as 1% of cell units counted. Dispersing bacterial mass indirectly to solution leads to homogeneous cell suspensions with repeatable cell concentrations. Viability of A. actinomycetemcomitans was also maintained when cells were suspended in water.

  • 18. Kisand, Veljo
    et al.
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences2003In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 54, no 2, p. 183-191Article in journal (Refereed)
    Abstract [en]

    The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tin and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers. (C) 2003 Elsevier Science B.V. All rights reserved.

  • 19.
    Koptina, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Volga State Univ Technol, Yoshkar Ola 424000, Russia..
    Strese, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Backlund, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Alsmark, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy. Natl Vet Inst SVA, Dept Virol Immunobiol & Parasitol, S-75651 Uppsala, Sweden..
    Challenges to get axenic cultures of Trichomonas spp.: A new approach in eradication of contaminants and maintenance of laboratory microbiological cultures2015In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 118, p. 25-30Article in journal (Refereed)
    Abstract [en]

    Contamination of microbiological and cell cultures is a major problem in many scientific and clinical laboratories as well as bioproduct manufacturers worldwide. In the current study we established a rapid (9 day) method to detect and eliminate fungal and bacterial contamination in cultures of the unicellular eukaryote Trichomonas spp. The developed method combines identification of the contaminating microorganisms using PCR and sequencing of the 16/18S regions followed by phylogenetic analysis. The next step was a phylogeny-guided selection of antibiotic treatments. We then used a two-step propidium iodide-resorufin assay to test the effect of selected antibiotics. The result was a quick and worthwhile purification of trichomonad laboratory cultures. Our workflow may also be implemented to obtain new isolates of trichomonads from clinical samples if initial broad-spectrum antibiotic therapy fails.

  • 20.
    Larsson, Marie C.
    et al.
    Linköping Univ Hosp.
    Lerm, Maria
    Linköping Univ.
    Angeby, Kristian
    Karolinska Univ Hosp ; Univ W Indies, Jamaica.
    Nordvall, Michaela
    Linköping Univ Hosp.
    Jureen, Pontus
    Publ Hlth Agcy Sweden.
    Schön, Thomas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Medicine and Optometry. Linköping Univ ; Kalmar County Hospital.
    A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 106, p. 146-150Article in journal (Refereed)
    Abstract [en]

    The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. (C) 2014 Elsevier B.V. All rights reserved.

  • 21.
    Larsson, Marie C
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Microbiology.
    Lerm, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Ängeby, Kristian
    Karolinska University Hospital, Hospital, Stockholm, Sweden; University of the West Indies, Kingston, Jamaica.
    Nordvall, Michaela
    Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Microbiology.
    Jureen, Pontus
    Public Health Agency of Sweden, Stockholm, Sweden.
    Schön, Thomas
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences. Kalmar County Hospital, Sweden; Linnaeus University, Kalmar, Sweden.
    A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 106, p. 146-150Article in journal (Refereed)
    Abstract [en]

    The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: greater than4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs.

  • 22. Lindstedt, Bjørn-Arne
    et al.
    Tham, Wilhelm
    Örebro University, School of Hospitality, Culinary Arts & Meal Science.
    Danielsson-Tham, Marie-Louise
    Örebro University, School of Hospitality, Culinary Arts & Meal Science.
    Vardund, Traute
    Helmersson, Seved
    Kapperud, Georg
    Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing2008In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 72, no 2, p. 141-148Article in journal (Refereed)
    Abstract [en]

    The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

  • 23.
    Liu, Yanling
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Elsholz, Bruno
    Fraunhofer Institute for Silicon Technology, Itzehoe, Germany.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Confirmative electric DNA array-based test for food poisoning Bacillus cereus2007In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 70, no 1, p. 55-64Article in journal (Refereed)
    Abstract [en]

    Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B. cereus pathogenicity, which results in diarrhea or emesis, were simultaneously evaluated on a 16-position electrical chip microarray. The DNA analyte preparation procedure comprising first 5 min of ultrasonic treatment, DNA extraction, and afterwards an additional 10 min sonication, was established as the most effective way of sample processing. No DNA amplification step prior to the analysis was included. The programmed assay was carried out within 30 min, once the DNA analyte from 10(8) bacterial cells, corresponding to one agar colony, was subjected to the assay. In general, this work represents a mature analytical way for DNA review. It can be used under conditions that require almost immediate results.

  • 24. Lu, Xuedong
    et al.
    Nie, Shuping
    Xia, Chengjing
    Huang, Lie
    He, Ying
    Wu, Runxiang
    Zhang, Li
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A rapid two-step algorithm detects and identifies clinical macrolide and beta-lactam antibiotic resistance in clinical bacterial isolates2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 102, p. 26-31Article in journal (Refereed)
    Abstract [en]

    Purpose: Aiming to identify macrolide and beta-lactam resistance in clinical bacterial isolates rapidly and accurately, a two-step algorithm was developed based on detection of eight antibiotic resistance genes. Methods: Targeting at genes linked to bacterial macrolide (msrA, ermA, ermB, and ermC) and beta-lactam (bla(TEM), bla(SHV), bla(CTX-N-1), bin(CTX-M-9)) antibiotic resistances, this method includes a multiplex real-time PCR, a melting temperature profile analysis as well as a liquid bead microarray assay. Liquid bead microarray assay is applied only when indistinguishable T-m profile is observed. Results: The clinical validity of this method was assessed on clinical bacterial isolates. Among the total 580 isolates that were determined by our diagnostic method, 75% of them were identified by the multiplex real-time PCR with melting temperature analysis alone, while the remaining 25% required both multiplex real-time PCR with melting temperature analysis and liquid bead microarray assay for identification. Compared with the traditional phenotypic antibiotic susceptibility test, an overall agreement of 81.2% (kappa = 0.614, 95% Cl = 0550-0.679) was observed, with a sensitivity and specificity of 87.7% and 73% respectively. Besides, the average test turnaround time is 3.9 h, which is much shorter in comparison with more than 24 h for the traditional phenotypic tests. Conclusions: Having the advantages of the shorter operating time and comparable high sensitivity and specificity with the traditional phenotypic test, our two-step algorithm provides an efficient tool for rapid determination of macrolide and beta-lactam antibiotic resistances in clinical bacterial isolates.  

  • 25. Lundén, K.
    et al.
    Eklund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Finlay, R.
    Stenlid, J.
    Asiegbu, F. O.
    Heterologous array analysis in Heterobasidion: Hybridisation of cDNA arrays with probe from mycelium of S, P or F-types2008In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 75, no 2, p. 219-224Article in journal (Refereed)
    Abstract [en]

    Because of the close relatedness between three species of Heterobasidion annosum (P type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type). we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.

  • 26.
    Marcinowska, Renata
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Mortiz, Thomas
    Surowiec, Izabella
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 87, no 1, p. 24-31Article in journal (Refereed)
    Abstract [en]

    Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600nm. At an appropriate density they are harvested by centrifugation, washed three times with NaCl solution and metabolites are extracted using methanol and a bead-mill. Dried extracts are methoxymated and derivatized with methyltrimethylsilyltrifluoroacetamide (MSTFA) then analyzed using gas chromatography coupled to time-of-flight mass spectrometry (GC-MS/TOF). Finally, patterns in the acquired data are examined by multivariate data modeling. This method enabled us to obtain reproducible metabolite profiles of Yersinia pseudotuberculosis, with about 25% compound identification, based on comparison with entries in available GC-MS libraries. To assess the potential utility of the method for comparative analysis of other bacterial species we analyzed cultures of Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and methicillin-sensitive Staphylococcus aureus (MSSA). Multivariate analysis of the acquired data showed that it was possible to differentiate the species according to their metabolic profiles. Our results show that the presented procedure can be used for metabolomic analysis of a wide range of bacterial species of clinical interest.

  • 27.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Olsson, Crister
    Nilsson, Isabelle
    Linköping University, Department of Medicine and Care. Linköping University, Faculty of Health Sciences.
    Grahn, Niclas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Benoni, Cecilia
    Ahrné, Siv
    Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis2005In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 63, no 3, p. 239-247Article in journal (Refereed)
    Abstract [en]

    Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis, the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens. © 2005 Elsevier B.V. All rights reserved.

  • 28.
    Monstein, Hans-Jürg
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Microbiology.
    Tärnberg, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Persis, Shohreh
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Johansson, Anders G
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Microbiology.
    Comparison of a capillary gel electrophoresis-based multiplex PCR assay and ribosomal intergenic transcribed spacer-2 amplicon sequencing for identification of clinically important Candida species2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 96, p. 81-83Article in journal (Refereed)
    Abstract [en]

    The performance of a commercially available Seegene Seeplex STI Master Panel 3 multiplex PCR for Candida species identification was compared with an internal transcribed spacer 2 (ITS2) PCR assay. We found that the Seeplex assay was specific for identification of C. albicans, C. krusei, C. parapsilosis, C. glabrata, C. tropicalis and C. dubliniensis.

  • 29.
    Periyannan Rajeswari, Prem Kumar
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Söderberg, Lovisa M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Yacoub, Alia
    Leijon, Mikael
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Multiple pathogen biomarker detection using an encoded bead array in droplet PCR2017In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 139, p. 22-28Article in journal (Refereed)
    Abstract [en]

    We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

  • 30.
    Rapp, Ellionor
    et al.
    Department of Laboratory Medicine, Clinical Microbiology, University Hospital, Örebro, Sweden.
    Samuelsen, Ørjan
    Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway; Microbial Pharmacology and Population Biology Research Group, Department of Pharmacy, The Arctic University of Norway (UiT), Tromsø, Norway.
    Sundqvist, Martin
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Detection of carbapenemases with a newly developed commercial assay using Matrix Assisted Laser Desorption Ionization-Time of Flight2018In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 146, p. 37-39Article in journal (Refereed)
    Abstract [en]

    This study evaluated the performance of the MBT STAR-Carba kit (Bruker Daltonics), to detect carbapenemase producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. in comparison with the RAPIDEC® CARBA NP test (BioMerieux). MBT STAR-Carba allowed the detection of carbapenemases in Enterobacteriaceae and P. aeruginosa.

  • 31. Riazi, Shadi
    et al.
    Dover, Sara
    KTH, School of Biotechnology (BIO).
    Turovskiy, Yevgeniy
    Chikindas, Michael L.
    Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides2007In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 71, no 1, p. 87-89Article in journal (Refereed)
    Abstract [en]

    BioRad's Rotofor (R) system has been frequently used for the purification of proteins and smaller pepticles such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor (R) isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances.

  • 32.
    Ryberg, Anna
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology.
    Olsson, Crister
    Malmö University Hospital.
    Ahrné, Siv
    Lund University.
    Monstein, Hans-Jürg
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology.
    Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, no 2, p. 183-188Article in journal (Refereed)
    Abstract [en]

    Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)5- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)5 and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)5 and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)5 and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.

  • 33. Sjöberg, Fei
    et al.
    Nowrouzian, Forough
    Rangel, Ignacio
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Hannoun, Charles
    MooreA, Edward
    Adlerberth, Ingegerd
    Wold, Agnes E.
    Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota2013In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 94, no 1, p. 37-46Article in journal (Refereed)
    Abstract [en]

    The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >10(6) CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.

  • 34.
    Storm, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Advani, Abdolreza
    Pettersson, Monica
    Hallander, Hans O
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Comparison of real-time PCR and pyrosequencing for typing Bordetella pertussis toxin subunit 1 variants2006In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 65, no 1, p. 153-158Article in journal (Refereed)
    Abstract [en]

    We describe two newly developed methods for rapid typing of the pertussis toxin subunit 1 gene (ptxS1). A real-time PCR assay based on hybridization probes and a Pyrosequencing assay were developed and the specificity, sensitivity, cost, hands-on time and post-assay data processing were compared to Sanger sequencing. Both methods enabled discrimination of all four allelic variants, correctly identified all ptxS1 alleles of 143 strains tested and proved suitable for large-scale screening of B. pertussis strains.

  • 35.
    Storm, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Gustafsson, Ingegerd
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Engstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Real-time PCR for pharmacodynamic studies of Chlamydia trachomatis2005In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 61, no 3, p. 361-367Article in journal (Refereed)
  • 36. Tavares, F
    et al.
    Sellstedt, Anita
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia2000In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 39, no 2, p. 171-178Article in journal (Refereed)
    Abstract [en]

    A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 pg protein per extraction procedure from exponentially growing cells, corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied. (C) 2000 Elsevier Science B.V. All rights reserved.

  • 37. Thierry, S.
    et al.
    Hamidjaja, R. A.
    Girault, G.
    Löfström, Charlotta
    DTU Technical University of Denmark, Denmark.
    Ruuls, R.
    Sylviane, D.
    A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis2013In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 95, no 3, p. 357-365Article in journal (Refereed)
    Abstract [en]

    Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2. ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. © 2013 Elsevier B.V.

  • 38.
    Ungphakorn, Wanchana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Malmberg, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Nielsen, Elisabet I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tängdén, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics2017In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 132, p. 69-75Article in journal (Refereed)
    Abstract [en]

    This study aimed to evaluate the potential of a new time-lapse microscopy based method (oCelloScope) to efficiently assess the in vitro antibacterial effects of antibiotics. Two E. con and one P. aeruginosa strain were exposed to ciprofloxacin, colistin, ertapenem and meropenem in 24-h experiments. Background corrected absorption (BCA) derived from the oCelloScope was used to detect bacterial growth. The data obtained with the oCelloScope were compared with those of the automated Bioscreen C method and standard time-kill experiments and a good agreement in results was observed during 6-24 h of experiments. Viable counts obtained at 1, 4, 6 and 24 h during oCelloScope and Bioscreen C experiments were well correlated with the corresponding BCA and optical density (OD) data. Initial antibacterial effects during the first 6 h of experiments were difficult to detect with the automated methods due to their high detection limits (approximately 105 CFU/mL for oCelloScope and 107 CFU/mL for Bioscreen C), the inability to distinguish between live and dead bacteria and early morphological changes of bacteria during exposure to ciprofloxacin, ertapenem and meropenem. Regrowth was more frequently detected in time-kill experiments, possibly related to the larger working volume with an increased risk of preexisting or emerging resistance. In comparison with Bioscreen C, the oCelloScope provided additional information on bacterial growth dynamics in the range of 105 to 107 CFU/mL and morphological features. In conclusion, the oCelloScope would be suitable for detection of in vitro effects of antibiotics, especially when a large number of regimens need to be tested.

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