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| 2. |
- Szponar, Bogumila, et al.
(författare)
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Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples
- 2002
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Ingår i: Journal of Microbiological Methods. - 1872-8359 .- 0167-7012. ; 50:3, s. 283-289
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Tidskriftsartikel (refereegranskat)abstract
- 3-Hydroxy fatty acids (3-OH FAs) of 10-18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria. These acids are used as chemical markers for determining endotoxin in environmental samples. The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples. Low levels (6.1 +/- 1.6-94.0 +/- 23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-fine rats. The levels were considerably higher (0.0-1.06 +/- 0.17 nmol/mg) in livers. The amounts of the 3-OH FAs did not differ between the two groups of rats. All analyses were made by gas chromatography-tandem mass spectrometry (GC-MSMS) for unequivocal identification. The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid beta-oxidation. (C) 2002 Elsevier Science B.V. All rights reserved.
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| 3. |
- 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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| 4. |
- 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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| 5. |
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| 6. |
- 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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| 7. |
- 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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| 8. |
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| 9. |
- 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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| 10. |
- 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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