| 1. |
- Anrup, Roland, et al.
(author)
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Centrala universitetsvärden hotas av bolagiseringsidén
- 2013
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In: Dagens nyheter. - 1101-2447.
-
Journal article (pop. science, debate, etc.)abstract
- Högskolestiftelser. Förslaget att driva svenska universitet i stiftelseform öppnar för bolagisering. Men det är ingen riktig utredning, utan en politisk pamflett utan eftertanke. Privatisering av universitet hotar både oberoendet, forskningskvaliteten och samhällsnyttan, skriver 36 forskare vid svenska högskolor och universitet.
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| 2. |
- Aprodu, Iuliana, et al.
(author)
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Advanced sample preparation for the molecular quantification of Staphylococcus aureus in artificially and naturally contaminated milk.
- 2011
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 145, s. 61-65
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Journal article (peer-reviewed)abstract
- Sample treatment is an essential element when using real-time PCR for quantification of pathogens directly on food samples. This study comparatively evaluated three different principles of sample treatment, i.e. immunomagnetic separation based on phage-derived cell wall binding molecules, matrix solubilization and flotation, in order to establish their suitability for quantifying low numbers of Staphylococcus aureus in milk. All three procedures succeeded to remove S. aureus from the milk matrix, either raw or pasteurized, and, as a result of the concentration of the target cells, minimized the effect of milk associated PCR inhibitors. Sample preparation based on immunomagnetic separation albeit of being user friendly, specific and rapid, failed to allow quantification of low and medium numbers (<10(4)CFU) of S. aureus. In a mastitic milk model cell wall binding domain (CBD)-based target cell extraction revealed results most closely matching those derived from culture-based quantification. Both matrix lysis and flotation allowed quantification of S. aureus at a level of 1-10 cells per ml. Both methods resulted in higher numbers of bacterial cell equivalents (bce) than plating could reveal. Since both methods harvest cells that have been subjected to either mechanical and chemical stresses before quantification, we concluded that the higher bce numbers resulted from a disaggregation of S. aureus clusters initially present in the inoculum. Conclusively, since likely each S. aureus cell of a toxigenic strain contributes to enterotoxin production, molecular quantification could provide an even more realistic impact assessment in outbreak investigations than plating does.
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| 3. |
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| 4. |
- Artin, Ingrid, et al.
(author)
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Effects of Carbon Dioxide on Growth of Proteolytic Clostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome
- 2010
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In: Applied and Environmental Microbiology. - 0099-2240. ; 76:4, s. 1168-1172
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Journal article (peer-reviewed)abstract
- The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.
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| 5. |
- Cao, Rong, et al.
(author)
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Elevated Enterotoxin A Expression and Formation in Staphylococcus aureus and its Association with Prophage Induction.
- 2012
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In: Applied and Environmental Microbiology. - 0099-2240. ; 78:14, s. 4942-4948
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Journal article (peer-reviewed)abstract
- Staphylococcus aureus strains producing the bacteriophage-encoded staphylococcal enterotoxin A (SEA) were divided into two groups, high- and low-SEA-producing strains, based on the amount of SEA produced. After growth under favorable conditions in batch cultures, ten of the 21 strains tested produced more than 1,000 ng/ml SEA and nine strains produced less than 10 ng/ml SEA; two enterotoxigenic strains, MRSA252 and Newman, produced intermediate levels of SEA (around 450 ng/ml). The differences in the production of SEA were found to be associated with the expression level of sea and whether they hosted the versions sea(1) or sea(2). Furthermore, differences in the nucleotide sequence in the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was correlated with the capacity for a massive increase in SEA levels by prophage induction as demonstrated using mitomycin C (MC). This was also confirmed by the occurrence of additional sea expression presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. Remarkably, the SEA level was increased by up to ten fold in some strains due to prophage induction. The low-SEA-producing group and the high-SEA-producing subgroup lacking phage activated sea transcription showed no increase in SEA formation after the addition of MC. This study demonstrates that sea expression in enterotoxigenic strains is correlated with the clonal lineage of sea-encoded phages. The high-SEA-producing group, and in particular the prophage inducible sea(1) group, may be more relevant in staphylococcal food poisoning than the low-SEA-producing group, mainly harboring sea(2).
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| 6. |
- Cao, Rong, et al.
(author)
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Inhibition kinetics of catabolic dehydrogenases by elevated moieties of ATP and ADP - implication for a new regulation mechanism in Lactococcus lactis
- 2010
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In: The FEBS Journal. - : Wiley. - 1742-464X. ; 277:8, s. 1843-1852
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Journal article (peer-reviewed)abstract
- ATP and ADP inhibit, in varying degrees, several dehydrogenases of the central carbon metabolism of Lactococcus lactis ATCC 19435 in vitro, i.e. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH). Here we demonstrate mixed inhibition for GAPDH and competitive inhibition for LDH and ADH by adenine nucleotides in single inhibition studies. The nonlinear negative co-operativity was best modelled with Hill-type kinetics, showing greater flexibility than the usual parabolic inhibition equation. Because these natural inhibitors are present simultaneously in the cytoplasm, multiple inhibition kinetics was determined for each dehydrogenase. For ADH and LDH, the inhibitor combinations ATP plus NAD and ADP plus NAD are indifferent to each other. Model discrimination suggested that the weak allosteric inhibition of GAPDH had no relevance when multiple inhibitors are present. Interestingly, with ADH and GAPDH the combination of ATP and ADP exhibits lower dissociation constants than with either inhibitor alone. Moreover, the concerted inhibition of ADH and GAPDH, but not of LDH, shows synergy between the two nucleotides. Similar kinetics, but without synergies, were found for horse liver and yeast ADHs, indicating that dehydrogenases can be modulated by these nucleotides in a nonlinear manner in many organisms. The action of an elevated pool of ATP and ADP may effectively inactivate lactococcal ADH, but not GAPDH and LDH, providing leverage for the observed metabolic shift to homolactic acid formation in lactococcal resting cells on maltose. Therefore, we interpret these results as a regulation mechanism contributing to readjusting the flux of ATP production in L. lactis.
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| 7. |
- Gronlund, Hugo, et al.
(author)
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Direct Detection Of Single-Nucleotide Polymorphisms In Bacterial DNA By SNPtrap
- 2011
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In: Preparative Biochemistry & Biotechnology. - : Informa UK Limited. - 1532-2297 .- 1082-6068. ; 41:2, s. 166-174
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Journal article (peer-reviewed)abstract
- A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technology that both is highly accurate and enables multiplexing. A current bottleneck for direct genome analyses by minisequencing, however, is the sensitivity, since minisequencing relies on linear signal amplification. Here, we present SNPtrap, which is a novel approach that combines the specificity and possibility of multiplexing by minisequencing with the sensitivity obtained by logarithmic signal amplification by polymerase chain reaction (PCR). We show a SNPtrap proof of principle in a model system for two polymorphic SNP sites in the Salmonella tetrathionate reductase gene (ttrC).
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| 8. |
- Grønlund, H., et al.
(author)
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Microarray-based genotyping of Salmonella : Inter-laboratory evaluation of reproducibility and standardization potential
- 2011
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 145:SUPPL. 1, s. S79-S85
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Journal article (peer-reviewed)abstract
- Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variation among different isolates in order to control pathogen-induced infections. Microarray technology is a promising diagnostic tool that provides genomic information on many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was designed in which the agreement of data from a DNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of the microarray technique as a first step towards standardization. The low-density array contains 281 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic marker genes associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer, wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. Critical parameters were randomized in a four-factorial experiment and statistical measures of inter-lab consistency and agreement were performed based on the kappa coefficient. A high level of agreement (kappa = 0.7-1.0) in microarray results was obtained even when employing different printing and hybridization facilities, different procedures for purifying genomic DNA and different wash buffers. However, less agreement (Kappa = 0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this parameter as being highly critical when transferring a standard microarray assay between laboratories. In conclusion, this study indicates that DNA microarray assays can be reproduced in at least two different facilities, which is a pre-requisite for the development of standard guidelines. © 2010 Elsevier B.V.
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| 9. |
- Hedman, Johannes, et al.
(author)
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Pre-PCR processing in bioterrorism preparedness : improved diagnostic capabilities for laboratory response networks
- 2013
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In: Biosecurity and bioterrorism. - : Mary Ann Liebert. - 1538-7135 .- 1557-850X. ; 11:S1, s. S87-S101
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Journal article (peer-reviewed)abstract
- Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification—that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis—so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.
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| 10. |
- Hedman, Johannes, et al.
(author)
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Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis
- 2010
-
In: Analytical Biochemistry. - - : Elsevier Inc.. - 0003-2697 .- 1096-0309. ; 405, s. 192-200
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Journal article (peer-reviewed)abstract
- The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substancespresent in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene salivastains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNApolymerase–buffer systems (Hedman et al., BioTechniques 47 (2009) 951–958). Here we show thatblending inhibitor-resistant DNA polymerase–buffer systems further increases the success rate of PCRfor various types of real crime scene samples showing inhibition. For 34 of 42 ‘‘inhibited” crime scenestains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq HotStart and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmedby analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerasesused separately for all tested sample types. When used separately, the performance of the DNApolymerases varied depending on the nature of the sample. The superiority of the blend is discussed interms of complementary effects and synergy between the DNA polymerase–buffer systems.
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| 11. |
- Hunt, Karen, et al.
(author)
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Classical enterotoxins of coagulase-positive Staphylococcus aureus isolates from raw milk and products for raw milk cheese production in Ireland
- 2012
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In: Dairy Science & Technology. - : Springer Science and Business Media LLC. - 1958-5586 .- 1958-5594. ; 92:5, s. 487-499
-
Journal article (peer-reviewed)abstract
- Toxin-producing Staphylococcus aureus can be present in raw milk and therefore in cheese made from raw milk. To determine the number and type of toxin producers in raw milk used for raw milk cheese production in Ireland, 117 samples of raw milk and related products from five raw milk suppliers, to four raw milk cheesemakers in the South of Ireland, were analysed for coagulase positive S. aureus. Enumeration, using ISO 688-2 and plating on Baird Parker Rabbit Plasma Fibrinogen selective agar showed samples were within limits set by EC regulations. Isolates (151 from 81 positive samples) were characterised for production of staphylococcal enterotoxins (SEs) SEA, SEB, SEC and SED by reverse passive latex agglutination (SET-RPLA) and by multiplex polymerase chain reaction for the sea, seb, sec, sed and see genes. The results showed 83.2% of the isolates did not contain the se genes or the toxin producing capability tested for. From only one supplier, 26 isolates contained the sec gene and produced SEC. Within these 26 isolates, there were only two PFGE types. One SEC-producing isolate showed no toxin production when grown in sterile 10% reconstituted skim milk at 10 degrees C and 12 degrees C for 96 and 74 h, respectively. Low concentrations of SEC were produced at 14 degrees C and 16 degrees C after 74 and 55 h, respectively. The results of this survey indicate that milk used for raw milk cheese production in Ireland poses a limited risk to public health, although further studies on occurrence of toxin producing S. aureus should be undertaken.
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| 12. |
- Laadan, Boaz, et al.
(author)
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Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants
- 2014
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13
-
Journal article (peer-reviewed)abstract
- Background: A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. Results: In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. Conclusions: We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.
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| 13. |
- Löfström, Charlotta, et al.
(author)
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Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR
- 2011
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 145:SUPPL. 1, s. S103-S109
-
Journal article (peer-reviewed)abstract
- To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4×102 to at least 2.2×107CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0±2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥6.1×108CFU/swab sample, but not by concentrations ≤6.1×106CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably. © 2010 Elsevier B.V.
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| 14. |
- Marta, Dora, et al.
(author)
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Extended staphylococcal enterotoxin D expression in ham products
- 2011
-
In: Food microbiology (Print). - : Elsevier BV. - 0740-0020 .- 1095-9998. ; 28:3, s. 617-620
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Journal article (peer-reviewed)abstract
- Staphylococcal enterotoxin D (SED) is one of the most frequently recovered enterotoxins in staphylococcal food poisoning (SFP) outbreaks. The expression and production of SED were investigated in three ham products, i.e. boiled ham, smoked ham and dry-cured Serrano ham incubated at room temperature for seven days. . Staphylococcus aureus was also, as a reference, grown in cultivation broth during optimal growth conditions for seven days. In boiled and smoked ham, continuous . sed expression was observed throughout the incubation period with a second increase in . sed expression found after five days of incubation. In smoked ham, nine times less SED per colony-forming unit of . S. aureus was detected than in boiled ham. In boiled ham, the SED levels unpredictably decreased after three days of incubation. In the Serrano ham, SED was detected after five days of incubation although . S. aureus growth was poor and . sed expression was too low to determine. After five days of incubation, all three products contained enough SED to cause SFP. These results show that the specific production levels of SED vary in the different ham products, and that toxin production was in part uncoupled from bacterial growth. © 2010 Elsevier Ltd.
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| 15. |
- Runquist, David, et al.
(author)
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Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae
- 2010
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 3
-
Journal article (peer-reviewed)abstract
- Background: Baker's yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results: In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions: Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.
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| 16. |
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| 17. |
- Schelin, Jenny, et al.
(author)
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Evaluation of pre-PCR processing approaches for enumeration of Salmonella enterica in naturally contaminated animal feed
- 2014
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In: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 116:1, s. 167-178
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Journal article (peer-reviewed)abstract
- Aims: Three pre-PCR processing strategies for the detection and/or quantification of Salmonella in naturally contaminated soya bean meal were evaluated. Methods and Results: Methods included: (i) flotation-qPCR [enumeration of intact Salmonella cells prior to quantitative PCR (qPCR)], (ii) MPN-PCR (modified most probable number method combined with qPCR) and (iii) qualitative culture enrichment PCR. The limit of quantification was 1·8 × 102 CFU g-1 (flotation-qPCR) and 0·02 MPN g-1 (MPN-PCR). Fifteen naturally contaminated Salmonella positive soya bean meal samples from one lot were analysed in parallel with the three methods, using 2·5, 50 and 25 g of feed, respectively, resulting in detection of Salmonella in 6, 15 and 9 bags. Enumeration resulted in 1·8 × 102-7·8 × 103 CFU g-1 (flotation-qPCR) and 0·024 to >5·2 MPN g-1 (MPN-PCR). Conclusions: Except for differences in methodology, results obtained with the three techniques could be due to the presence of nonculturable Salmonella and/or a heterogeneous distribution of Salmonella in the material. Significance and Impact of the Study: The evaluated methods provide different possibilities to assess the prevalence of Salmonella in feed, together with the numbers of culturable, as well as nonculturable cells, and can be applied to generate data to allow more accurate quantitative microbial risk assessment for Salmonella in the feed chain. © 2013 The Society for Applied Microbiology.
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| 18. |
- Schelin, Jenny, et al.
(author)
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The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment.
- 2011
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In: Virulence. - : Informa UK Limited. - 2150-5608 .- 2150-5594. ; 2:6, s. 580-592
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Research review (peer-reviewed)abstract
- The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety.
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| 19. |
- Skorupa Parachin, Nadia, et al.
(author)
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Flotation as a tool for indirect DNA extraction from soil
- 2010
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 87:5, s. 1927-1933
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Journal article (peer-reviewed)abstract
- Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 mug DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.
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| 20. |
- van Niel, Ed, et al.
(author)
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The potential of biodetoxification activity as a probiotic property of Lactobacillus reuteri.
- 2012
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 152, s. 206-210
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Journal article (peer-reviewed)abstract
- Previous work on the metabolism of Lactobacillus reuteri ATCC 55730 anticipated a variability in the use of organic electron acceptors as a means to relieve metabolic redox problems. Therefore, investigations focusing on this unique metabolism of L. reuteri may reveal a basis for new probiotic properties. For instance, L. reuteri may use reactive aldehydes and ketones as electron acceptors to balance their redox metabolism, which opens the possibility to exploit this bacterium for in vivo bioreduction of deleterious compounds in the gastrointestinal tract (GIT). Herein we demonstrate that L. reuteri ATCC 55730 cultures on glucose are able to use furfural (1g/L), and hydroxymethylfurfural (HMF) (0.5g/L), as electron acceptors. The former enhances the growth rate by about 25% and biomass yield by 15%, whereas the latter is inhibitory. Furfural is stoichiometrically reduced to furfuryl alcohol by the culture. The conversion of furfural had no effect on the flux distribution between the simultaneously operating phosphoketolase and Embden-Meyerhof pathways, but initiated a flux to acetate production. In addition to furfural and HMF, cellular extracts showed potential to reoxidize NADH and/or NADPH with acrolein, crotonaldehyde, and diacetyl, indicating that conversion reactions take place intracellularly, however, utilization mechanisms for the latter compounds may not be present in this strain. The strain did not reduce other GIT-related reactive compounds, including acrylamide, glyoxal, and furan.
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| 21. |
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| 22. |
- Zeaki, Nikoleta, et al.
(author)
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Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage.
- 2014
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 182, s. 44-50
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Journal article (peer-reviewed)abstract
- Three Staphylococcus aureus strains representing different alleles of the Siphoviridae prophage-encoded enterotoxin A (SEA) gene, including two high-SEA-producing strains and one low-SEA-producing strain were studied to investigate sea expression and SEA formation on a frankfurter type of sausage. The effect of lactic acid, an antimicrobial compound used as a preservative in food, was also investigated on the same product. All three strains were grown on pork sausages at 15°C for 14days in the presence or absence of lactic acid (1 or 2% v/v). Growth, sea mRNA expression and SEA formation were regularly monitored and compared between non-treated and treated sausages. For all experiments performed, the extracellular SEA formation significantly differed between the high- and low-SEA-producing strains, although growth and viability were overall the same. For the low producer (Sa51), the accumulated amount of extracellular SEA formed after 14days was close to the detection limit (less than 1ng/g) in all conditions; while Sa21 and Sa17, the two high-producing strains, formed 250±25.37ng/g and 750±82.65ng/g in non-treated sausage and 150±75.75ng/g and 300±83.89ng/g when treated with 1% lactic acid, respectively, after 14days. Sausages treated with 2% lactic acid followed the same pattern as above, but with an extended lag phase to 4days and reduced levels of enterotoxin formed for all strains. The difference in the level of SEA between the two high-producing strains is most likely due to the different clonal lineages of the sea-encoded Siphoviridae phages where induction of the prophage potentially could be the reason for higher production of SEA in one of the lines. Furthermore, a prolonged expression of sea gene in the two high-producing strains was observed during the entire incubation period, while the sea expression was under the detection limit in the low-producing strain. This study indicates that the high-SEA-producing strains, especially the strains with the putative capacity of prophage induction, could be more relevant in food safety aspects than low-producing type of strains on pork sausage.
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| 23. |
- Zeidan, Ahmad, et al.
(author)
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Stable coexistence of two Caldicellulosiruptor species in a de novo constructed hydrogen-producing co-culture
- 2010
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 9
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Journal article (peer-reviewed)abstract
- Background Mixed culture enrichments have been used frequently for biohydrogen production from different feedstock. In spite of the several advantages offered by those cultures, they suffer poor H2 yield. Constructing defined co-cultures of known H2 producers may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved. Results The extreme thermophiles Caldicellulosiruptor saccharolyticus DSM 8903 and C. kristjanssonii DSM 12137 were combined in a co-culture for H2 production from glucose and xylose in a continuous-flow stirred tank reactor. The co-culture exhibited a remarkable stability over a period of 70 days under carbon-sufficient conditions, with both strains coexisting in the system at steady states of different dilution rates, as revealed by speciesspecific quantitative PCR assays. The two strains retained their ability to stably coexist in the reactor even when glucose was used as the sole growth-limiting substrate. Furthermore, H2 yields on glucose exceeded those of either organism alone under the same conditions, alluding to a synergistic effect of the two strains on H2 production. A maximum H2 yield of 3.7 mol (mol glucose)-1 was obtained by the co-culture at a dilution rate of 0.06 h-1; a higher yield than that reported for any mixed culture to date. A reproducible pattern of population dynamics was observed in the co-culture under both carbon and non-carbon limited conditions, with C. kristjanssonii outgrowing C. saccharolyticus during the batch start-up phase and prevailing at higher dilution rates. A basic continuous culture model assuming the ability of C. saccharolyticus to enhance the growth of C. kristjanssonii could mimic the pattern of population dynamics observed experimentally and provide clues to the nature of interaction between the two strains. As a proof, the cell-free growth supernatant of C.saccharolyticus was found able to enhance the growth of C. kristjanssonii in batch culture through shortening its lag phase and increasing its maximum biomass concentration by ca. 18%. Conclusions This study provides experimental evidence on the stable coexistence of two closely related organisms isolated from geographically-distant habitats under continuous operation conditions, with the production of H2 at high yields. An interspecies interaction is proposed as the reason behind the remarkable ability of the two Caldicellulosiruptor strains to coexist in the system rather than only competing for the growth-limiting substrate.
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