| 1. |
- Andersson, Ulrika, et al.
(author)
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Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes
- 2005
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In: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020. ; 28:3, s. 187-195
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Journal article (peer-reviewed)abstract
- Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons encoding the proteins and enzymes crucial for catabolism of lactose, maltose and threhalose revealed an obvious unity in operon organisation. The local regulator of each operon was located in a divergent transcriptional direction to the rest of the operon including the transport protein-encoding genes. Furthermore, in all three operons a catabolite responsive element (CRE) site was detected inbetween the gene encoding the local regulator and one of the genes encoding ! sugar transport protein. It is evident that regardless of type of transport system and catabolic enzymes acting upon lactose, maltose and trehalose, respectively, Lc. lactis shows unity in both operon organisation and regulation of these catabolic operons. This knowledge was further extended to other catabolic operons in Lc. lactis and the two related bacteria Lactobacillus plantarum and Listeria monocytogenes. Thirty-nine catabolic operons responsible for degradation of sugars and sugar alcohols in Lc. lactis, Lb. plantarum and L. monocytogenes were investigated and the majority of those possessed the same organisation as the lactose, maltose and trehalose operons of Lc. lactis. Though, the frequency of CRE sites and their location varied among the bacteria. Both Lc. lactis and Lb. plantarum showed CRE sites in direct proximity to genes coding for proteins responsible for sugar uptake. However, in, L. monocytogenes CRE sites were not frequently found and not in the vicinity of genes encoding transport proteins, suggesting a more local mode of regulation of the catabolic operons found and/or the use of inducer control in this bacterium. © 2004 Elsevier GrnbH. All rights reserved.
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| 2. |
- Artin, Ingrid, et al.
(author)
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Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum type E
- 2008
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 74:8, s. 2391-2397
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Journal article (peer-reviewed)abstract
- Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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| 3. |
- Artin, Ingrid, et al.
(author)
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First case of type E wound botulism diagnosed using real-time PCR.
- 2007
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In: Journal of Clinical Microbiology. - 1098-660X. ; 45:11, s. 3589-3594
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Journal article (peer-reviewed)abstract
- Wound botulism is a growing problem among injecting drug users. The condition is often difficult to diagnose, with laboratory confirmation in only 50% of the cases. Here we present a real-time PCR-based method for the diagnosis of wound botulism caused by Clostridium botulinum. The assay includes an internal amplification control which is amplified simultaneously with the genes encoding neurotoxin types A, B, and E. This method was used to detect the first case of wound botulism in an injecting drug user in Sweden. In addition, to the best of our knowledge, this is the first reported case of wound botulism caused by C. botulinum type E.
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| 4. |
- Dai, JY, et al.
(author)
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Conformational cycling in beta-phosphoglucomutase catalysis: Reorientation of the beta-D-glucose 1,6-(bis) phosphate intermediate
- 2006
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:25, s. 7818-7824
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Journal article (peer-reviewed)abstract
- Activated Lactococcus lactis beta-phosphoglucomutase (beta PGM) catalyzes the conversion of beta-D-glucose 1-phosphate (beta G1P) derived from maltose to beta-D-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-1), K-m = 49 +/- 4 mu M for the substrate, beta G1P and K-m = 6.5 +/- 0.7 mu M for the activator beta-D-glucose 1,6-bisphosphate (beta G1,6bisP). The observed transient accumulation of [C-14]beta G1,6bisP (12% at similar to 0.1 s) in the single turnover reaction carried out with excess beta PGM (40 mu M) and limiting [C-14]beta G1P (5 mu M) and beta G1,6bisP (5 mu M) supported the role of beta G1,6bisP as a reaction intermediate in the conversion of the, G1P to G6P. Single turnover reactions of [C-14]beta G1,6bisP with excess, beta PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the beta G1,6bisP binds to the active site in two different orientations (one positioning the C(1) phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6) phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with beta PGM, [C-14]beta G1P, and unlabeled beta G1,6bisP demonstrated complete exchange of label to the beta G1,6bisP during the catalytic cycle. Thus, the reorientation of the beta G1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of beta G1P suggest that the reorientation and phosphoryl transfer from beta G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of beta G1,6bisP to beta G1P plus G6P was examined to evidence a significant stabilization of beta PGM aspartyl phosphate.
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| 5. |
- de Vin, Filip, et al.
(author)
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Molecular and biochemical analysis of the galactose phenotype of dairy Streptococcus thermophilus strains reveals four different fermentation profiles
- 2005
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In: Applied and Environmental Microbiology. - 0099-2240. ; 71:7, s. 3659-3667
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Journal article (peer-reviewed)abstract
- Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related.
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| 6. |
- Eriksson, John, et al.
(author)
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Comparison of genotyping methods by application to Salmonella livingstone strains associated with an outbreak of human salmonellosis
- 2005
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 104:1, s. 93-103
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Journal article (peer-reviewed)abstract
- During 2000 and 2001, an outbreak of human salmonellosis occurred in Sweden and Norway, caused by Salmonella livingstone. In this study, the genotypic differences between three strains obtained from food sources during the outbreak, two human strains and 27 more or less unrelated strains were analysed, using the three methods; automated ribotyping, pulsed field get electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD). Each method was evaluated regarding its discriminatory ability, reproducibility and typeability. Simpson's discriminatory index calculated for each method was 0.556 for automated ribotyping, 0.766 for PFGE and 0.236 for RAPD. The reproducibility, defined as the minimum similarity between individual replicates in a cluster analysis, was 96% for automated ribotyping and PFGE, and 90% for RAPD. All the strains were typeable with each method. When combining results for the three genotyping methods, it was found that RAPD did not increase the discriminatory index and was therefore excluded from further analysis. Using a combination of the results obtained from ribotyping and PFGE (D = 0.855), two strains that had been isolated from feed factories during 1998 were shown to be identical to the outbreak strain, indicating a possible route of contamination due to a clone of Salmonella livingstone persisting in feed producing facilities. No connection to poultry was established. (c) 2005 Elsevier B.V. All rights reserved.
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| 7. |
- Hedman, Johannes, et al.
(author)
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Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles
- 2009
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In: BIOTECHNIQUES. - : Eaton Publishing. - 0736-6205 .- 1940-9818. ; 47:5, s. 951-958
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Journal article (peer-reviewed)abstract
- DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.
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| 8. |
- Jannasch, Patric, et al.
(author)
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Kamratgranskning av muntliga gruppresentationer: pålitlighet och effekter på studenternas lärande
- 2009
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In: Proceeding från Den 2:a Utvecklingskonferensen för Sveriges ingenjörsutbildningar.
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Conference paper (peer-reviewed)abstract
- Det finns goda skäl till att engagera studenterna i kamratbedömning vid både formativ och summativ examination. Vi har under kursen Molekylär Cellbiologi för Ekosystemvetare vid LTH undersökt studenternas förmåga att bedöma muntliga projektredovisningar och jämfört den med lärarnas. Under kursen genomförde studenterna ett litteraturprojekt där en muntlig gruppredovisning med efterföljande kamratdiskussion och försvar bedömdes och poängsattes av en opponerande studentgrupp och ett lärarlag. Vår undersökning visade att studenternas poängbedömning stämde väl överens med lärarlagets, både med avseende på medelvärde och spridning. Små skillnader visade sig i bedömningen av ”faktainnehåll” och ”samarbete” där lärarlaget gav ett något mer positivt omdöme, och i bedömning av ”försvar” där studentgrupperna gav lite högre poäng. En undersökning av studenternas attityder och upplevelser i samband med kamratgranskningen visade att presenterande studenter upplevde sig rättvist bedömda, men att några granskande studenter kände lite obehag och osäkerhet inför kamratbedömningen. Vidare upplevde både lärare och studenter att lärandet om ämnet, lärandeprocessen och examinationsprocessen gynnades av kamratgranskningen och kamratdiskussionerna. Sammanfattningsvis visade våra resultat att kamratgranskningen fungerade väl, och att studenterna behöver utbildning och tydlig information om bedömningskriterier och motiven för granskningsprocessen så att de kan känna sig trygga, framför allt i granskarrollen.
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| 9. |
- Jannasch, Patric, et al.
(author)
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Kamratgranskning och kamratdiskussion vid bedömning av muntliga gruppresentationer: pålitlighet och effekter på studenternas lärande
- 2009
-
In: Proceedings från Lunds universitets andra utvecklingskonferens.
-
Conference paper (peer-reviewed)abstract
- Det finns goda skäl till att engagera studenterna i deras egen bedömning vid både formativ och summativ examination. Vi har under kursen Molekylär Cellbiologi för Ekosystemvetare vid LTH undersökt studenternas förmåga att bedöma muntliga projektredovisningar och jämfört den med lärarnas. Under kursen genomförde studenterna ett projekt där en muntlig gruppredovisning med efterföljande kamratdiskussion och försvar bedömdes och poängsattes av en opponerande studentgrupp och ett lärarlag. Vår undersökning visade att studenternas poängbedömning stämde väl överens med lärarlagets, både med avseende på medelvärde och spridning. Små skillnader visade sig i bedömningen av ”faktainnehåll” och ”samarbete” där lärarlaget gav ett något mer positivt omdöme, och i bedömning av ”försvar” där studentgrupperna gav lite högre poäng. En undersökning av studenternas attityder och upplevelser i samband med kamratgranskningen visade att presenterande studenter upplevde sig rättvist bedömda, men att några granskande studenter kände lite obehag och osäkerhet inför kamratbedömningen. Vidare upplevde både lärare och studenter att lärandet om både ämnet, lärandeprocessen och bedömningsprocessen gynnades av kamratgranskningen och kamratdiskussionerna. Sammanfattningsvis visade våra resultat att kamratgranskningen fungerade väl, och att studenterna behöver utbildning och tydlig information om bedömningskriterier och motiven för granskningsprocessen så att de kan känna sig trygga, framför allt i granskarrollen.
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| 10. |
- Laadan, Boaz, et al.
(author)
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Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Saccharomyces cerevisiae.
- 2008
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In: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:3, s. 191-198
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Journal article (peer-reviewed)abstract
- We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from the industrial Saccharomyces cerevisiae strain TMB3000 that mediates the NADH-dependent reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis-hydroxymethylfuran. The co-factor preference distinguished this alcohol dehydrogenase from the previously reported NADPH-dependent S. cerevisiae HMF alcohol dehydrogenase Adh6. The amino acid sequence revealed three novel mutations (S109P, L116S and Y294C) that were all predicted at the vicinity of the substrate binding site, which could explain the unusual substrate specificity. Increased biomass production and HMF conversion rate were achieved in a CEN.PK S. cerevisiae strain overexpressing the mutated ADH1 gene. Copyright (c) 2008 John Wiley & Sons, Ltd.
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| 11. |
- Löfström, Charlotta, et al.
(author)
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Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp enterica serovar Senftenberg strains isolated from feed mills
- 2006
-
In: Veterinary Microbiology. - : Elsevier BV. - 0378-1135 .- 1873-2542. ; 114:3-4, s. 345-351
-
Journal article (peer-reviewed)abstract
- In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 +/- 16.9% and 72.3 +/- 12.9% for Taq to 91.6 +/- 7.5% and 90.9 +/- 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD, types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE
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| 12. |
- Löfström, Charlotta, et al.
(author)
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Validation of a Diagnostic PCR Method for Routine Analysis of Salmonella spp. in Animal Feed Samples
- 2008
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In: Food Analytical Methods. - : Springer Science and Business Media LLC. - 1936-9751 .- 1936-976X. ; 1:1, s. 23-27
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Journal article (peer-reviewed)abstract
- As a part of a validation study, a comparative study of a PCR method and the standard culture-based method NMKL-71, for detection of Salmonella, was performed according to the validation protocol from the Nordic validation organ for validation of alternative microbiological methods (NordVal) on 250 artificially or naturally contaminated animal feed samples. The PCR method is based on culture enrichment in buffered peptone water followed by PCR using the DNA polymerase Tth and an internal amplification control. No significant difference was found between the two methods. The relative accuracy, relative sensitivity and relative specificity were found to be 96.0, 97.3, and 98.8%, respectively. PCR inhibition was observed for rape seed samples. For the acidified feed samples, more Salmonella-positive samples were found with the PCR method compared to the NMKL method. This study focuses on the growing demand for validated diagnostic PCR methods for routine analysis of animal feed and food samples to assure safety in the food production chain.
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| 13. |
- Runquist, David, et al.
(author)
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Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae.
- 2009
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:1, s. 123-130
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Journal article (peer-reviewed)abstract
- The glucose/xylose facilitator Gxf1 from Candida intermedia was expressed in the recombinant xylose-fermenting Saccharomyces cerevisiae strain TMB 3057. The new strain, TMB 3411, displayed approximately two times lower K (m) for xylose transport compared to a control strain not expressing Gxf1. In aerobic batch cultivation, the specific growth rate was significantly higher at low xylose concentration, 4 g/L, when Gxf1 was expressed, whereas it remained unchanged at high xylose concentration, 40 g/L. Similarly, in aerobic-xylose-limited chemostat culture, the Gxf1-expressing strain consumed more xylose than the control strain at low dilution rates (low xylose concentration), whereas the situation was reversed at higher dilution rates (high xylose concentration). Also, under anaerobic conditions, the Gxf1-expressing strain showed faster xylose uptake and ethanol formation at low substrate concentrations. The results are discussed in relation to previous observations, which suggested that transport controlled xylose utilization in recombinant xylose-utilizing S. cerevisiae only at low xylose concentrations.
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| 14. |
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| 15. |
- Svensson, Malin, et al.
(author)
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Altered nucleotide sugar metabolism in Streptococcus thermophilus interferes with nitrogen metabolism
- 2007
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 113:2, s. 195-200
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Journal article (peer-reviewed)abstract
- Exopolysaccharide (EPS)-producing Streptococcus thermophilus strains have attracted interest recently, since the EPSs act as natural viscosifiers and texture enhancers of fermented foods. We have previously reported that the low level of EPS production by S. thermophilus LY03 could be improved by altering the activities of enzymes in the central carbon metabolism involved in the nucleotide sugar metabolism. In this study, we observed a reduced growth in milk for the strains with increased UDP-glucose pyrophosphorylase (GalU) activity together with either enhanced phosphoglucomutase activity, and/or enhanced activity of the Leloir enzymes. Rapid growth of these mutants in milk could be restored by the addition of four specific amino acids, i.e. Glu, His, Met, and Val. This amino acid requirement was confirmed in a defined medium. Furthermore, the P-31 NMR spectra showed higher levels of the GalU reactants pyrophosphate (PPi) and UDP-glucose in the engineered strain, TMB 6013, compared to the parent strain, LY03. These products plus Glu and the GalU reactant UTP are known to be involved in the nitrogen regulatory system in many bacteria. Thus, these results suggest that the reaction catalyzed by GalU is connected to the nitrogen demand of these engineered strains. (c) 2006 Elsevier B.V. All rights reserved.
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| 16. |
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| 17. |
- Velasco, Susana, et al.
(author)
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Environmental factors influencing growth of and exopolysaccharide formation by Pediococcus parvulus 2.6
- 2006
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 111:3, s. 252-258
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Journal article (peer-reviewed)abstract
- Natural exopolysaccharides (EPSs) from food-grade lactic acid bacteria have potential for development and exploitation as food additives and functional food ingredients with both health and economic benefits. In this study, we have examined the physiological capacity of EPS production in Pediococcus parvulus 2.6. EPS formation by P. parvulus 2.6 was found to be linked to biomass yields, provided that glucose was not limiting. Higher biomass yields and EPS productions were obtained when cultures were pH-controlled at pH 5.2. Various compounds have been tested for their influence on growth rate and EPS formation. Of those, only glucose (up to 75 g 1(-1)), ethanol (up to 4.9%, w/v) and glycerol (up to 6.6%, w/v) had positive effects on EPS production. EPS production was not directly linked to growth, because its production continued in the stationary phase provided that glucose was present. According to an empirical model, the growth of R parvulus 2.6 was completely inhibited by 58.9 +/- 18.1 gl(-1) lactate. Lactate, the sole fermentation product, was suggested to affect growth by chelation of manganese. The organism grew in an apparent linear fashion due to this imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg l(-1) in the medium. The excretion of Mn2+ upon depletion of glucose indicated that maintenance of the high Mn2+ gradient over the cell membrane is an energy requiring process. EPS production was increased from 0.12 gl(-1) to 4.10 gl(-1) in an improved medium that is based on the results from this study. (c) 2006 Elsevier B.V. All rights reserved.
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| 18. |
- Wolffs, Petra, et al.
(author)
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Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR
- 2005
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In: Applied and Environmental Microbiology. - 0099-2240. ; 71:10, s. 5759-5764
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Journal article (peer-reviewed)abstract
- Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 X 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 X 10(3) CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20 degrees C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% +/- 9% and 23% +/- 4%, respectively, at 4 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.
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| 19. |
- Wolffs, Petra, et al.
(author)
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Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells
- 2005
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In: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 60:3, s. 315-323
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Journal article (peer-reviewed)abstract
- Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized. (C) 2004 Published by Elsevier B.V.
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| 20. |
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| 21. |
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| 22. |
- Årsköld, Emma, et al.
(author)
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Environmental influences on exopolysaccharide formation in Lactobacillus reuteri ATCC 55730.
- 2007
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 116:1, s. 159-167
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Journal article (peer-reviewed)abstract
- Lactobacillus reuteri is known to produce exopolysaccharides (EPS), which have the potential to be used as an alternative biothickener in the food industry. In this study, the effect of several environmental conditions on the growth and EPS production in the L. reuteri strain ATCC 55730 was determined. The expression of the corresponding reuteransucrase gene, gtfO, was investigated over time and the results indicated that the expression increased with growth during the exponential phase and subsequently decreased in the stationary phase. Fermentation with glucose and/or sucrose as carbon and energy source revealed that gtfO was constitutively expressed and that the activity profile was independent of the sugar source. In the applied ranges of parameter values, temperature and pH were the most important factors for EPS formation and only temperature for growth. The best EPS yield, 1.4 g g(-1) CDW, was obtained at the conditions 37 degrees C, pH 4.5 and 100 g l(-1) sucrose, which were close to the estimated optimal conditions: pH 4.56 and 100 g l(-1) sucrose. No EPS formation could be detected with glucose. In addition, no direct connection between the expression and the activity of reuteransucrase could be established. Finally, the strain ATCC 55730 was benchmarked against 14 other L. reuteri strains with respect to EPS production from sucrose and abilities to metabolise sucrose, glucose and fructose. Eight strains were able to produce glucan and a corresponding glucansucrase gene was confirmed for each of them. (c) 2007 Elsevier B.V. All rights reserved.
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| 23. |
- Årsköld, Emma, et al.
(author)
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Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis
- 2008
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In: Journal of Bacteriology. - 0021-9193. ; 190:1, s. 206-212
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Journal article (peer-reviewed)abstract
- Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro 31P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.
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