| 1. |
- Bal, Karol, et al.
(författare)
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Structure of muramic acid TMS derivative mass spectrum's base ion (m/z=185) used for quantification of bacterial peptidoglycan.
- 2002
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Ingår i: Journal of Microbiological Methods. - 1872-8359. ; 48:2-3, s. 267-270
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Tidskriftsartikel (refereegranskat)abstract
- The use of trimethylsilyl (TMS)-derivatisation for determining muramic acid in environmental and clinical samples by gas chromatography-mass spectrometry provides high detection sensitivity; however, questions have been raised as concerns the chemical structure of the entity giving the strong signal of m/z 185. In the present communication we present evidence that this entity results from the formation of a lactam structure of muramic acid upon derivatisation.
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| 2. |
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| 3. |
- Bjerketorp, J., et al.
(författare)
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Rapid lab-on-a-chip profiling of human gut bacteria
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 72:1, s. 82-90
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Brolund, Alma, et al.
(författare)
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Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 82:3, s. 229-233
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBLM) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBLM are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. Material and methods: Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmo University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. Results and discussion: The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated. (C) 2010 Elsevier B.V. All rights reserved.
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| 5. |
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| 6. |
- Bunikis, Ignas, et al.
(författare)
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Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi.
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 86:2, s. 243-7
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Chandler, Jeffrey C., et al.
(författare)
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Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces
- 2020
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Ingår i: Journal of Microbiological Methods. - : ELSEVIER. - 0167-7012 .- 1872-8359. ; 172
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Tidskriftsartikel (refereegranskat)abstract
- A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 mu g/mL) and vancomycin (8 mu g/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 10(0) and 10(4) CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.
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| 8. |
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| 9. |
- Crocetti, Greg, et al.
(författare)
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An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH)
- 2006
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 65:1, s. 194-201
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence in situ hybridisation (FISH) is a common and popular method used to investigate microbial populations in natural and engineered environments. DNA oligonucleoticle probes require accurate determination of the optimal experimental conditions for their use in FISH Oligonucleotides targeting the rRNA of methanogenic Archaea at various taxonomic levels have previously been published, although when applied in FISH, no optimisation data has been presented In this study, 3000 Euryarchaeota 16S rRNA gene sequences were phylogenetically analysed and previously published oligonucleoticles were evaluated for target group accuracy. Where necessary, modifications were introduced or new probes were designed. The updated set of probes was optimised for use in FISH for a more accurate detection of methanogenic Archaea. (c) 2005 Elsevier B.V. All rights reserved.
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| 10. |
- Dahlén, Gunnar, 1944, et al.
(författare)
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Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique.
- 2015
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Ingår i: Journal of microbiological methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 110, s. 68-77
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Tidskriftsartikel (refereegranskat)abstract
- The reproducibility and reliability of quantitative microbiological assessments using the DNA-DNA hybridization "checkerboard method" (CKB) were assessed. The data originated from 180 chronic periodontitis patients, who were enrolled in a clinical trial and sampled at baseline, and 3 and 12m post-therapy. The samples were divided into two portions allowing evaluation of reproducibility. In total, 531 samples were analyzed in a first run, using standard bacterial preparations of cells and 513 samples were accessible for analysis in the second, using standards based on purified DNA from the species. The microbial probe panel consisted of periodontitis marker bacteria as well as non-oral microorganisms. Three different ways of quantifying and presenting data; the visual scoring method, VSM, the standard curve method, SCM, and the percent method, PM, were compared. The second set of analyses based on the use of standard preparations of pure DNA was shown to be more consistent than the first set using standards based on cells, while the effect of storage time per se up to 2.5y seemed to be marginal. The best reproducibility was found for Tannerella forsythia, irrespective of quantification technique (Spearman's rho=0.587, Pearson's r≥0.540). The percent method (PM) based on percent of High Standard (10(6) cells) was more reliable than SCM based on a linear calibration of the High Standard and a Low Standard (10(5) cells). It was concluded that the reproducibility of the CBK method varied between different bacteria. High quality and pure specific DNA whole genomic probes and standards may have a stronger impact on the precision of the data than storage time and conditions.
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| 11. |
- Danilov, Roman, et al.
(författare)
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Comparison of usefulness of three types of artificial substrata (glass, wood and plastic) when studying settlement patterns of periphyton in lakes of different trophic status
- 2001
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Ingår i: Journal of Microbiological Methods. - 0167-7012 .- 1872-8359. ; 45:3, s. 167-170
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Dierikx, Cindy, et al.
(författare)
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A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae
- 2022
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 193
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Tidskriftsartikel (refereegranskat)abstract
- The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.
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| 13. |
- Dopson, Mark, et al.
(författare)
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First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships.
- 2004
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Ingår i: J Microbiol Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 58:3, s. 297-302
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 14. |
- Ek, H., et al.
(författare)
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Determination of N-15-labeled ammonium and total nitrogen in plant and fungal systems using mass-spectrometry
- 1990
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Ingår i: Journal of Microbiological Methods. - 1872-8359. ; 11:3-4, s. 169-176
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Tidskriftsartikel (refereegranskat)abstract
- A selected ion-monitoring method to measure 15N-labelled ammonia in biological samples was improved to simplify sample handling, to obviate interference from ammonia due to the decomposition of glutamine and to allow the determination of total N. Ammonia is derivatized with pentafluorobenzoylchloride to yield pentafluorobenzamide which is analysed by gas chromatography/mass spectrometry after clean-up using disposable silicic acid columns. The sensitivity achieved when operating in the negative ion-chemical ionization mode was somewhat higher than when using electron-impact ionization. Use of methyl amine as an internal standard improved the accuracy and precision of the measurements. The method was applied to samples taken from an intact ectomycorrhizal system fed with ISN-labelled ammonium and used to determine patterns of N assimilation into ammonium, free amino acids and macromolecular compounds.
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| 15. |
- Eriksson, Ronnie, et al.
(författare)
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Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout
- 2009
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 78:2, s. 195-202
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 16. |
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| 17. |
- Fatima, Masoom, et al.
(författare)
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Application of novel bacterial consortium for biodegradation of aromatic amine 2-ABS using response surface methodology
- 2020
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 174
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Tidskriftsartikel (refereegranskat)abstract
- There is a strong need to develop purification methods for textile industrial wastewater containing toxic azo dyes. The reductive cleavage of azo dyes can be made by anaerobic bacteria, but the products of aromatic amines require an aerobic process. In this study a novel bacterial dye degrading consortium (DDC) of five isolated strains identified with 16S rRNA sequence: Proteus mirabilis (KR732288), Bacillus anthracis (KR732289), Enterobacter hormaechei (KR732290), Pseudomonas aeruginosa (KR732293) and Serratia rubidaea (KR732296) were used to aerobically decompose metabolite 2-aminobenxenesulfonic acid (2-ABS), as a model compound. The effect of three variables: temperature (28-42 degrees C), pH (5.0-8.0) and initial concentration of 2-ABS (5-40 ppm) was investigated in terms of degradation and chemical oxygen demand (COD) removal. Central composite design matrixand response surface methodology (RSM) were used for experimental design to evaluate theinteraction of the three process variables. The results show that up to 95% degradation and COD 90% removal are possible at optimal values of 32.4 ppm 2-ABS, pH 6.6 and a temperature of 35.7 degrees C. The theoretical response variables predicted by the developed RSM model was supported the experimental results. The optimized degradation of 2-ABS and COD removal were further confirmed by UV-HPLC analysis.
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| 18. |
- Fellström, Claes, et al.
(författare)
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Identification and genetic fingerprinting of Brachyspira species
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 72:2, s. 133-140
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Tidskriftsartikel (refereegranskat)abstract
- Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intennedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.
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| 19. |
- Ferrando, R, et al.
(författare)
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3-Hydroxy fatty acids in saliva as diagnostic markers in chronic periodontitis
- 2005
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 62:3, s. 285-291
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Tidskriftsartikel (refereegranskat)abstract
- Saturated straight- and branched-chain 3-hydroxy fatty acids (3-OH FAs) of 10-18 carbon chain lengths were determined in saliva from 27 individuals with chronic periodontitis and 18 healthy individuals by using gas chromatography-tandem mass spectrometry. Of the 14 different 3-OH FAs detected, 3-OH-C-i17:0 was the most abundant in the periodontitis samples while 3-OH-C-14:0 was the most abundant in the healthy individuals. Considering the relative percentages of 3-OH-C-12:0, 3-OH-C-14:0, 3OH-C-i17:0, and 3-OH-C-17:0, 95.6% of all cases were correctly classified as healthy individuals or periodontitis patients by means of discriminant analysis. The sensitivity, specificity, positive predictive value and negative predictive value of 3-OH FA analysis in diagnosing peridontitis were, respectively, 0.92, 1.00, 1.00, and 0.90. The results indicate that 3-OH FA analysis of saliva samples is a useful diagnostic method in chronic periodontitis.
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| 20. |
- Forsgren, Eva
(författare)
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An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens
- 2021
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 184
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Tidskriftsartikel (refereegranskat)abstract
- Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on & nbsp;a & nbsp;panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded
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| 21. |
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| 22. |
- Fridlund, Jimmy, et al.
(författare)
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A microbiological method for determining serum levels of broad spectrum β-lactam antibiotics in critically ill patients
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 129, s. 23-27
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 23. |
- Gantelius, Jesper, et al.
(författare)
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A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 82:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 24. |
- Gantner, S, et al.
(författare)
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Novel primers for 16S rRNA-based archaeal community analyses in environmental samples
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 84:1, s. 12-18
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 25. |
- Gorokhova, Elena, et al.
(författare)
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A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy
- 2012
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 89:3, s. 216-221
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 26. |
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| 27. |
- Hellman, Maria, et al.
(författare)
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Survey of bromodeoxyuridine uptake among environmental bacteria and variation in uptake rates in a taxonomically diverse set of bacterial isolates
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 86, s. 376-378
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Tidskriftsartikel (refereegranskat)abstract
- Incorporation of 5-Bromo-2'-Deoxyuridine (BrdU) into DNA can be used to target replicating bacteria in the environment, but differential uptake capacity is a potential bias. Among 23 bacterial isolates commonly found in soils, most took up BrdU, but at up to 10-fold different cell-specific rates. Combined with results from an in silico analysis of 1000 BrdU-labeled 16S rRNA gene sequences, our results demonstrate a BrdU uptake bias with no apparent relationship between taxa affiliation and ability to incorporate BrdU. (C) 2011 Elsevier B.V. All rights reserved.
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| 28. |
- Håkansson, Sebastian
(författare)
-
Survival and phosphate solubilisation activity of desiccated formulations of Penicillium bilaiae and Aspergillus niger influenced by water activity
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 150, s. 39-46
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Tidskriftsartikel (refereegranskat)abstract
- The impact of formulation and desiccation on the shelf life of phosphate (P)-solubilising microorganisms is often under-studied, particularly relating to their ability to recover P-solubilisation activity. Here, Penicilllium bilaiae and Aspergillus niger were formulated on vermiculite (V) alone, or with the addition of protectants (skimmed milk (V + SM) and trehalose (V + T)), and on sewage sludge ash with (A + N) and without nutrients (A), and dried in a convective air dryer. After drying, the spore viability of P. bilaiae was greater than that of A. niger. V formulations achieved the highest survival rates without being improved by the addition of protectants. P. bilaiae formulated on V was selected for desiccation in a fluidised bed dryer, in which several temperatures and final water activities (aw) were tested. The highest spore viability was achieved when the formulation was dried at 25 degrees C to a final aw > 0.3. During three months' storage, convective air dried formulations were stable for both strains, except in the presence of skimmed milk for P. bilaiae which saw a decrease in spore viability. In the fluidised bed-dried formulations, when aw > 0.3, the loss in viability was higher, especially when stored at 20 degrees C, than at aw < 0.1. P-solubilisation activity performed on ash was preserved in most of the formulations after desiccation and storage. Overall, a low drying temperature and high final aw positively affected P. bilaiae viability, however a trade-off between higher viability after desiccation and shelf life should be considered. Further research is needed to optimise viability over time and on more sustainable carriers.
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| 29. |
- Ibrahim, Victor, et al.
(författare)
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Enzymatic monitoring of lignin and lignin derivatives biooxidation.
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 120, s. 53-55
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Tidskriftsartikel (refereegranskat)abstract
- Lignin oxidation was enzymatically monitored by measuring methanol released during the reaction. The methanol was oxidized to formaldehyde and hydrogen peroxide, and the latter used to oxidize ABTS to a product measured spectrophotometrically. The efficiency was comparable to the commonly used gas chromatography method. The assay was fast and inexpensive.
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| 30. |
- Ihalin, R, et al.
(författare)
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16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.
- 2006
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 65:3, s. 417-424
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 31. |
- Isaksson, Jenny, et al.
(författare)
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Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 127, s. 214-218
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 32. |
- Jagadish, Anupama, et al.
(författare)
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Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pebrine disease in Bombyx mori silkworm, based on the β-tubulin gene
- 2021
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 186
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Tidskriftsartikel (refereegranskat)abstract
- “Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.
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| 33. |
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| 34. |
- Kalle Kossio, Elena
(författare)
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External and semi-internal controls for PCR amplification of homologous sequences in mixed templates
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 95, s. 285-294
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Tidskriftsartikel (refereegranskat)abstract
- In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control: therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. (C) 2013 Elsevier B.V. All rights reserved.
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| 35. |
- Karched, M, et al.
(författare)
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A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 68:1, s. 46-51
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- 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.
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| 36. |
- Kisand, Veljo, et al.
(författare)
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Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences
- 2003
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Ingår i: Journal of Microbiological Methods. - 0167-7012 .- 1872-8359. ; 54:2, s. 183-191
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 37. |
- Koptina, Anna, et al.
(författare)
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Challenges to get axenic cultures of Trichomonas spp. : A new approach in eradication of contaminants and maintenance of laboratory microbiological cultures
- 2015
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 118, s. 25-30
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 38. |
- Larsson, Marie C, et al.
(författare)
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A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability
- 2014
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 106, s. 146-150
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 39. |
- Linder, Tomas
(författare)
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A standardized toolkit for genetic engineering of CTG Glade yeasts
- 2018
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 144, s. 152-156
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a series of synthetic constructs suitable to genetically manipulate a broad range of yeast species belonging to the fungal CTG Glade. This molecular toolbox notably allows heterologous gene expression, single or dual fluorescence labeling and construction of luciferase-expressing strains for bioluminescence imaging.
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| 40. |
- Lindstedt, Bjørn-Arne, et al.
(författare)
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Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 72:2, s. 141-148
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 41. |
- Liu, Yanling, 1974-, et al.
(författare)
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Confirmative electric DNA array-based test for food poisoning Bacillus cereus
- 2007
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 70:1, s. 55-64
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 42. |
- Lu, Xuedong, et al.
(författare)
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A rapid two-step algorithm detects and identifies clinical macrolide and beta-lactam antibiotic resistance in clinical bacterial isolates
- 2014
-
Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 102, s. 26-31
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 43. |
- Lundén, K., et al.
(författare)
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Heterologous array analysis in Heterobasidion : Hybridisation of cDNA arrays with probe from mycelium of S, P or F-types
- 2008
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 75:2, s. 219-224
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 44. |
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| 45. |
- Løvdal, Trond, et al.
(författare)
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Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 85:2, s. 164-169
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Tidskriftsartikel (refereegranskat)abstract
- The combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) significantly overestimated the fraction of viable Listeria innocua as compared to plate counts and confocal fluorescence microscopy. Our data imply that PMA-qPCR must be used with caution as an analytical tool for the differentiation between viable and dead bacteria.
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| 46. |
- Marcinowska, Renata, et al.
(författare)
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Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 87:1, s. 24-31
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 47. |
- Markowicz, Pawel, et al.
(författare)
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The surface emissions trap: A new approach in indoor air purification.
- 2012
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 91:2, s. 290-294
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Tidskriftsartikel (refereegranskat)abstract
- A new device for stopping or reducing potentially irritating or harmful emissions from surfaces indoors is described. The device is a surface emissions trap prototype and consists of an adsorbent sheet with a semipermeable barrier surrounded by two thin nonwoven layers. The trap may be applied directly at the source of the emissions e.g. at moisture-affected floors and walls, surfaces contaminated by chemical spills etc. This results in an immediate stop or reduction of the emitting pollutants. The trap has a very low water vapor resistance thus allowing drying of wet surfaces. In laboratory experiments typically 98% reduction of air concentrations of volatile organic compounds and a virtually total reduction of mold particle-associated mycotoxins was observed. The surface emissions trap may represent a convenient and efficient way of restoring indoor environments polluted by microbial and other moisture-associated emissions.
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| 48. |
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| 49. |
- Monstein, Hans-Jurg, 1946-, et al.
(författare)
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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 analysis
- 2005
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 63:3, s. 239-247
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Tidskriftsartikel (refereegranskat)abstract
- 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. (c) 2005 Elsevier B.V. All rights reserved.
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| 50. |
- Nestor, David, 1992-, et al.
(författare)
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Evaluation of the FilmArray (TM) Meningitis/Encephalitis panel with focus on bacteria and Cryptococcus spp
- 2019
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 157, s. 113-116
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Molecular methods provide fast and accurate detection of both bacteria and viruses in the cerebrospinal fluid (CSF) causing infection in the central nervous system (CNS). In the present study we evaluated the bacterial detection performance of the fully automated FilmArray (TM) Meningitis/Encephalitis (ME) panel (bioMerieux) by comparing it with culture and multiplexed in-house PCR. Methods: Three sample types were analysed; Contrived samples with known bacterial/fungal concentration (n = 29), clinical samples from patients with verified cause of CNS infection (n = 17) and external quality assessment (EQA) samples (n = 11). Another six samples were purposely prepared with multiple targets to evaluate multiplex capacity. Results: The FilmArray (TM) had a slightly higher limit of detection for Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes and Streptococcus agalactiae compared to in-house PCR methods but performed equal or better when compared to culture. The FilmArray (TM) ME panel detected the expected pathogen in 17 of 17 clinical samples and yielded detection of three additional viruses of which one was confirmed with comparator techniques. All but one of the EQA samples were correctly detected. Conclusions: The results of this study are promising and the FilmArray (TM) ME panel could add to the diagnostic algorithm in CNS-infections. However, the limit of detection for the important pathogens N. meningitidis and S. pneumoniae could be improved.
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