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Martin Wagner - One of the best experts on this subject based on the ideXlab platform.

  • Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC)
    Food Analytical Methods, 2012
    Co-Authors: Karin Frühwirth, Martin Wagner, Sabine Fuchs, Patrick Mester, Peter Rossmanith
    Abstract:

    Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in Food Pathogen detection. Food Pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes . A Δ- prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during real-time PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ- prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua . The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain ( P  ≤ 0.05).

  • A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for Food Pathogen quantification.
    Journal of food protection, 2011
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in Food Pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

  • The challenge to quantify Listeria monocytogenes– a model leading to new aspects in molecular biological Food Pathogen detection
    Journal of applied microbiology, 2010
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    Summary In this work, we discuss the latest insights concerning advantages and disadvantages and the nature of microbiological and molecular methods for quantitative Food Pathogen detection. The assessment of molecular methods must be brought on a basis that considers the nature of molecular methods and their underlying mechanism. A potential approach to setting up the development, validation and structure of an analytical chain is presented based on quantitative real-time PCR (qPCR). This is analysed exemplary on the basis of recent work using the model organism Listeria monocytogenes. Several prerequisites for successful quantitative detection of this Pathogen will be discussed. In particular, sample preparation, controls for all methodical steps and the validation of the core assay qPCR are addressed, which constitute the basis for a reliable analytical detection chain for molecular biological Pathogen detection from Food. Microbiological methods are analysed based on growth of the single cell, which is the fundament of these traditional methods.

  • Aspects of systems theory in the analysis and validation of innovative molecular-biological based Food Pathogen detection methods
    Trends in Food Science and Technology, 2010
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    Molecular-biological methods for Food analysis comprise a detection chain consisting of sample preparation, target purification and detection assays. Given this systemic character systems theory provides basis for discussion of alternative approaches for specification and validation of Pathogen detection methods using testing methods derived from other scientific areas. Since the underlying black box mechanism of microbiological methods does not meet the same systemic criteria, microbiological and molecular-biological methods cannot be compared one-to-one in Food Pathogen detection. This work describes structure and strategy of an alternative approach for analysis and validation to promote broad range implementation of molecular-biological methods in Food Pathogen detection.

Peter Rossmanith - One of the best experts on this subject based on the ideXlab platform.

  • Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC)
    Food Analytical Methods, 2012
    Co-Authors: Karin Frühwirth, Martin Wagner, Sabine Fuchs, Patrick Mester, Peter Rossmanith
    Abstract:

    Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in Food Pathogen detection. Food Pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes . A Δ- prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during real-time PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ- prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua . The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain ( P  ≤ 0.05).

  • A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for Food Pathogen quantification.
    Journal of food protection, 2011
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in Food Pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

  • The challenge to quantify Listeria monocytogenes– a model leading to new aspects in molecular biological Food Pathogen detection
    Journal of applied microbiology, 2010
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    Summary In this work, we discuss the latest insights concerning advantages and disadvantages and the nature of microbiological and molecular methods for quantitative Food Pathogen detection. The assessment of molecular methods must be brought on a basis that considers the nature of molecular methods and their underlying mechanism. A potential approach to setting up the development, validation and structure of an analytical chain is presented based on quantitative real-time PCR (qPCR). This is analysed exemplary on the basis of recent work using the model organism Listeria monocytogenes. Several prerequisites for successful quantitative detection of this Pathogen will be discussed. In particular, sample preparation, controls for all methodical steps and the validation of the core assay qPCR are addressed, which constitute the basis for a reliable analytical detection chain for molecular biological Pathogen detection from Food. Microbiological methods are analysed based on growth of the single cell, which is the fundament of these traditional methods.

  • Aspects of systems theory in the analysis and validation of innovative molecular-biological based Food Pathogen detection methods
    Trends in Food Science and Technology, 2010
    Co-Authors: Peter Rossmanith, Martin Wagner
    Abstract:

    Molecular-biological methods for Food analysis comprise a detection chain consisting of sample preparation, target purification and detection assays. Given this systemic character systems theory provides basis for discussion of alternative approaches for specification and validation of Pathogen detection methods using testing methods derived from other scientific areas. Since the underlying black box mechanism of microbiological methods does not meet the same systemic criteria, microbiological and molecular-biological methods cannot be compared one-to-one in Food Pathogen detection. This work describes structure and strategy of an alternative approach for analysis and validation to promote broad range implementation of molecular-biological methods in Food Pathogen detection.

Remco Kort - One of the best experts on this subject based on the ideXlab platform.

  • multiparameter viability assay for stress profiling applied to the Food Pathogen listeria monocytogenes f2365
    Applied and Environmental Microbiology, 2011
    Co-Authors: Andreas Nocker, Athina Esveldamanatidou, Jos M B M Van Der Vossen, Frank H J Schuren, Martien P M Caspers, Roy Christiaan Montijn, Remco Kort
    Abstract:

    A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This Food-borne Pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.

  • multiparameter viability assay for stress profiling applied to the Food Pathogen listeria monocytogenes f2365
    Applied and Environmental Microbiology, 2011
    Co-Authors: Andreas Nocker, Athina Esveldamanatidou, Frank H J Schuren, Martien P M Caspers, Roy Christiaan Montijn, Jos M B M Van Der Vossen, Remco Kort
    Abstract:

    A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This Food-borne Pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters. © 2011, American Society for Microbiology.

Barbara C. A. Dowds - One of the best experts on this subject based on the ideXlab platform.

  • Acid stress in the Food Pathogen Bacillus cereus
    Journal of applied microbiology, 2002
    Co-Authors: N. Browne, Barbara C. A. Dowds
    Abstract:

    Aims: The Pathogen Bacillus cereus, which is associated with a number of Foods including dairy products, was studied for its response to acid stress during the exponential phase. Methods and Results:Bacillus cereus was found to adapt to acid stress (pH 4·6) when pre-exposed to a non-lethal, inducing pH of 6·3 or to inducing concentrations of heat, ethanol, salt or hydrogen peroxide. Cells were found to maintain their internal pH at a higher level than the external acid pH and adapted cells had a higher internal pH than unadapted cells. A constitutive acid-sensitive mutant that was also heat- and ethanol-sensitive was found to be capable of high levels of adaptation despite its lack of induction of proteins induced in the wild type by exposure to moderate pH (6·3) values. Conclusions: A number of proteins were found to be underexpressed in the mutant compared with the wild type at pH 6·3, including some with homology to ribosomal proteins and to the sporulation regulator RapK, while one differentially expressed band contained two proteins, one of which was homologous to the competence regulator CodY. Significance and Impact of the Study: The work has implications for the processing of B. cereus-associated Foods by acidification. The linked developmental processes of stationary phase, sporulation and possibly competence appear to be involved in the response to acid stress.

  • Heat and salt stress in the Food Pathogen Bacillus cereus
    Journal of applied microbiology, 2001
    Co-Authors: N. Browne, Barbara C. A. Dowds
    Abstract:

    Aims: The effects of stresses imposed on bacterial contaminants during Food processing and treatment of packaging material were evaluated on the Food Pathogen Bacillus cereus. Methods and Results: Conditions were established which allowed the cells to adapt to heat, ethanol and hydrogen peroxide stresses, but not to osmotic shock. Cross protection between stresses indicated a clear hierarchy of resistance with salt protecting against hydrogen peroxide, which protected against ethanol, which protected against heat shock. The cultures were shown to be most sensitive to heat, ethanol and oxidative stress at mid-exponential phase and to become resistant at stationary phase. Adaptive levels of stressor were found to induce synthesis of general stress and stress-specific proteins and differential accumulation of proteins was demonstrated between heat- or salt-stressed and unstressed cells. Conclusions: Sequencing revealed that a number of glycolytic enzymes were regulated by heat and osmotic shocks and that the chaperone GroEL was induced by heat shock. Significance and Impact of the Study: The implications of the physiological data in designing storage and processing conditions for Food are discussed. The identification of stress-regulated proteins reveals a clear role for glycolysis in adaptation to heat shock and osmotic stress.

Andreas Nocker - One of the best experts on this subject based on the ideXlab platform.

  • multiparameter viability assay for stress profiling applied to the Food Pathogen listeria monocytogenes f2365
    Applied and Environmental Microbiology, 2011
    Co-Authors: Andreas Nocker, Athina Esveldamanatidou, Jos M B M Van Der Vossen, Frank H J Schuren, Martien P M Caspers, Roy Christiaan Montijn, Remco Kort
    Abstract:

    A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This Food-borne Pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.

  • multiparameter viability assay for stress profiling applied to the Food Pathogen listeria monocytogenes f2365
    Applied and Environmental Microbiology, 2011
    Co-Authors: Andreas Nocker, Athina Esveldamanatidou, Frank H J Schuren, Martien P M Caspers, Roy Christiaan Montijn, Jos M B M Van Der Vossen, Remco Kort
    Abstract:

    A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This Food-borne Pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters. © 2011, American Society for Microbiology.