Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54
    Hayrapetyan, Hasmik - \ 2017
    Wageningen University. Promotor(en): T. Abee, co-promotor(en): M.N. Nierop Groot. - Wageningen : Wageningen University - ISBN 9789463431194 - 181
    microorganisms - bacillus cereus - food contamination - biofilms - foodborne pathogens - abiotic conditions - sporulation - micro-organismen - bacillus cereus - voedselbesmetting - biofilms - voedselpathogenen - abiotiek - sporulatie

    Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired biofilms can be formed by spoilage and pathogenic microorganisms in food production environments. Bacillus cereus is a foodborne human pathogen able to cause two types of food poisoning, emetic and diarrheal. B. cereus can persist in factory environments in the form of biofilms, which can become a source of food contamination. This thesis adds to the knowledge about (a)biotic factors and conditions that affect B. cereus biofilm formation, including the effect of type of substratum such as polystyrene and stainless steel, with the latter supporting the highest biofilm formation for all tested strains including two reference strains and 20 food isolates. The ability of B. cereus to use a variety of iron sources was subsequently studied in these 22 strains and linked to the genes encoding iron transport systems present in the respective genomes, revealing significant diversity in the capacity to use complex and non-complex iron sources for growth and biofilm formation. For spore forming Bacilli, biofilm formation and sporulation are two intertwined cellular processes and studies in wet and dry (air-exposed) biofilms revealed differences in sporulation rate and efficacy, with biofilm-derived spores showing higher heat resistance than their planktonic counterparts. Additionally, comparative phenotype and transcriptome analysis of B. cereus wild type and a Sigma 54 deletion mutant provided insight into the pleiotropic role of this transcriptional regulator in B. cereus biofilm formation and physiology in general. Taken together, this knowledge improves our understanding of the biofilm lifecycle of this notorious food-borne human pathogen and provides clues which can help to reduce the domestication of this microorganism in production environments.

    Bacillus cereus spore damage recovery and diversity in spore germination and carbohydrate utilisation
    Warda, Alicja K. - \ 2016
    Wageningen University. Promotor(en): Tjakko Abee, co-promotor(en): Masja Nierop Groot. - Wageningen : Wageningen University - ISBN 9789462579262 - 157
    bacterial spores - bacillus cereus - heat treatment - damage - microbiological techniques - bacteriële sporen - bacillus cereus - warmtebehandeling - schade - microbiologische technieken

    Bacterial spores are extremely robust survival vehicles that are highly resistant towards environmental stress conditions including heat, UV radiation and other stresses commonly applied during food production and preservation. Spores, including those of the toxin-producing food-borne human pathogen Bacillus cereus, are ubiquitously present in a wide range of environmental niches such as soil, plant rhizosphere, intestinal tract of insects and animals, and it is virtually impossible to prevent contamination at the primary production level. Heat treatments are conventionally applied in food processing to reduce the microbial load of food products, however, to comply with consumer desire for products with higher sensory and nutritional values, the treatment intensity may become milder. Consequently, subpopulations of spores may emerge that are sublethally damaged rather than inactivated conceivably causing quality and safety issues following repair and outgrowth. In this thesis, a functional genomics approach was used in combination with subpopulation and single spore analysis to identify factors involved in recovery of heat damaged spores, and to link B. cereus genotypes to nutrient-induced germination capacity and carbohydrate utilisation capacity.

    Using comparative analysis of B. cereus ATCC 14579 wild type and targeted mutants, putative damage repair factors were identified such as putative transcriptional regulator CdnL, that supported recovery of spores in a range of conditions including model foods. The majority of identified genes encoding putative damage repair factors appeared to be unique for B. cereus group members. This novel information on spore recovery adds to further insights in versatility of survival strategies of B. cereus.

    Different types of foods may contain different types and levels of nutrients including amino acids and carbohydrates, that can affect spore germination capacity and subsequent outgrowth performance of vegetative B. cereus cells. Nutrient germinants present in food products can trigger specific germinant receptors (GRs) located in the spore inner membrane leading to spore germination, a critical step before growth resumes. Combined analysis of genotypes and nutrient-induced germination phenotypes using high throughput flow cytometry analysis at the level of individual spores, revealed substantial diversity in germination capacity with a subset of strains showing a very weak germination response even in nutrient-rich media containing high levels of amino acids. Phylogenetically, these B. cereus strains grouped in subgroup IIIA encompassing strains containing pseudogenes or variants of some of the Ger clusters and two strains containing the recently identified SpoVA2mob transposon, that induced heat resistance with concomitant reduced germination response in Bacillus subtilis spores. The same B. cereus isolates were also used to link genotypes with carbohydrate utilisation clusters present on the genomes, and this revealed representatives of subgroup IIIA to lack specific carbohydrate utilisation clusters (starch, glycogen, aryl beta-glucosides; salicin, arbutin and esculin) suggesting a reduced capacity to utilise plant-associated carbohydrates for growth. Since these B. cereus subgroup IIIA representatives contain host-associated carbohydrate utilisation gene clusters and a subset of unique Ger clusters, their qualification as poor germinators may require revision following assessment of spore germination efficacy using host-derived compounds as germinants.

    The research described in this thesis has added novel insights in B. cereus capacity to cope with spore damage and provided novel overviews of the distribution and putative functionality of (sub)clusters of GRs and carbohydrate utilisation clusters. Knowledge on spore damage repair, germination and metabolism capacity adds to further understanding of B. cereus ecology including niche occupation and transmission capacity.

    Werkgroep Fytobacteriologie : verslag van de bijeenkomst op 3 september 2015 : dead or alive
    Wolf, J.M. van der; Overbeek, L.S. van - \ 2015
    Gewasbescherming 46 (2015)4. - ISSN 0166-6495 - p. 124 - 124.
    plantenziekteverwekkende bacteriën - bacteriologie - werkgroepen - plantenziektekunde - voedselveiligheid - biologische technieken - bacillus cereus - komkommerbontvirus - nuttige organismen - plant pathogenic bacteria - bacteriology - working groups - plant pathology - food safety - biological techniques - bacillus cereus - cucumber green mottle mosaic virus - beneficial organisms
    Het thema van deze werkgroepbijeenkomst ‘dead or alive’ was al tijdens een eerder werkgroepoverleg gekozen. Het kunnen onderscheiden van dode en levende bacteriën is belangrijk, zowel voor pathogene bacteriën als voor ‘beneficials’, maar het onderscheid is vaak moeilijk te maken.
    Impact of sorbic acid and other mild preservation stresses on germination and outgrowth of Bacillus cereus spores
    Melis, C.C.J. van - \ 2013
    Wageningen University. Promotor(en): Tjakko Abee, co-promotor(en): Masja Nierop Groot. - Wageningen : Wageningen University - ISBN 9789461737755 - 138
    bacillus cereus - sorbinezuur - behoud - kieming - kiemremmers - groei - voedselbewaring - voedselmicrobiologie - bacillus cereus - sorbic acid - preservation - germination - germination inhibitors - growth - food preservation - food microbiology

    Weak organic acids such as sorbic acid, lactate, and acetic acid are widely used by the food industry as preservatives to control growth of micro-organisms. With the current trend towards milder processing of food products, opportunities arise for spore-forming spoilage and pathogenic microorganisms such as Bacillus cereus, that may survive the use of milder heating regimes. Dormant spores produced by B. cereus can survive processing conditions and their subsequent outgrowth increases the risk of premature spoilage and food safety issues. As a consequence, the use of additional preservation hurdles, such as acidification with weak organic acid additives to ensure the quality and safety of a product is important. Sorbic acid is widely used as an antimicrobial compound because of its strong inhibitory properties against bacteria and other spoilage organisms. Its effectivity has also been ascribed to its hydrophobic character, resulting in an additional mode of action not observed with other less lipophilic organic acids such as lactic acid and acetic acid. In this project the impact of sorbic acid on spore germination and outgrowth was studied at transcriptome level and was linked to the distinct phenotypic responses observed for spores exposed to different levels of sorbic acid. The various stages of spore germination and outgrowth could be recognized by distinct gene expression profiles representing either the germination phase, transition state between germination and outgrowth or outgrowing cells, respectively. A subset of genes was specifically expressed in sorbic acid-exposed germinating spores and included functions related to cell envelope, (multi) drug transporters and amino acid metabolism. At high concentrations of sorbic acid (3mM of the undisssociated form, HSA), nutrient-induced germination of B. cereus ATCC 14579 spores was completely blocked. This blockage was shown to be reversible and could be bypassed by known non-nutrient triggers that activate spores in a receptor-independent way, pointing to a possible interference of HSA with the signaling event between germinant receptors and proposed SpoVA-channels, possibly by accumulation into the spore’s inner membrane. Additional experiments with other inhibiting compounds, including organic acids and their structurally similar alcohol counterparts, showed that the lipophilic properties are an important determinant of its efficacy to block germination. Building on current knowledge on the interaction of germination-relevant protein clusters, we discuss a hypothetical model on the mode of action of sorbic acid and other short-chain lipophilic compounds in germination inhibition of B. cereus spores. In addition to the interference or even blockage of germination, sorbic acid may increase outgrowth heterogeneity when applied at lower concentrations (0.25-1.5 mM) that still allow outgrowth. The first stages of outgrowth were shown to specifically occur heterogeneously when spores were exposed to multiple stresses simultaneously. Heterogeneity effects were most pronounced for combined stress-effects where heat-treated spores were also exposed to low pH stress. Under these conditions, a large subpopulation of spores was delayed between initial germination and swelling and further outgrowth. For the food producing industry, it would be desirable to have reliable parameters to predict the behavior of surviving spores in a food product. Data presented in this thesis show that germination rate is not a good predictor for outgrowth heterogeneity when applied as a single indicator. In conclusion, the work described in this thesis strive to obtain a better understanding on the impact that preservation stresses, including (sorbic) acid stress, have on the germination and outgrowth (heterogeneity) of B. cereus spores. The results obtained in this project may contribute to the rational design of new concepts for improved food preservation and safety.

    Bacillus cereus: emetic toxin production and gamma hypothesis for growth
    Biesta-Peters, E.G. - \ 2011
    Wageningen University. Promotor(en): Marcel Zwietering; Leon Gorris, co-promotor(en): Martine Reij. - [S.l.] : S.n. - ISBN 9789085859932 - 192
    bacillus cereus - groei - bacteriële toxinen - voedselbewaring - voedselveiligheid - bacillus cereus - growth - bacterial toxins - food preservation - food safety

    Bacillus cereus is a food spoilage microorganism and a pathogen. Growth of B. cereus can

    be prevented or delayed by adding growth limiting compounds to the food product or

    by altered storage conditions. Combinations of growth limiting factors can show synergy,

    or be multiplicative without synergy (gamma hypothesis). For food safety management,

    it is important to understand if combinations are synergistic or not, to avoid making faildangerous

    or overly fail-safe predictions. Therefore, the aim of this PhD project was to

    validate the gamma hypothesis for specific combinations of hurdles commonly used in

    food production. Since the relationship between growth and toxin production of B. cereus

    is little understood, a second aim was to investigate the production of the emetic toxin

    cereulide in more detail. Several new lines of research were set-up to deliver on these

    aims. For growth data collection to quantify hurdle effects and to study combinations

    of hurdles, the relative rate to detection method was found to be preferred over two

    other methods evaluated. The gamma hypothesis was validated for combinations of pH

    and undissociated acid. For combinations of pH and water activity lowering solutes, the

    gamma hypothesis could neither be validated nor rejected. The validity of the gamma

    hypothesis appeared to be dependent on the models chosen for the single hurdle effects,

    which are subsequently combined into the gamma model. A systematic way of model

    selection is therefore advocated. Investigating cells in the transition from lag phase (λ)

    to exponential growth phase, it was found that trends in physiological processes could

    be observed for different culture conditions, independent of the duration of λ. Esterase

    activity and electron transport chain activity were found to be useful quantitative markers

    for this transition phase. A new method to produce synthetic cereulide showing biological

    activity was developed, allowing accurate quantification of cereulide in samples. The

    use of valinomycin as a standard, the current procedure, underestimates the amount of

    cereulide by approximately 10 %. Considering the onset of emetic toxin production, LCMS

    analysis of B. cereus F4810/72 grown in BHI showed that cereulide production does

    not start before cells are in mid to late stationary phase, although significant variation

    was noted possibly related to variability in the growth parameters maximum specific

    growth rate (μmax) and λ. Addition of salt to the growth medium delayed the production

    of cereulide. This research has been able to deliver several new insights and tools that are

    useful for food safety management of the emetic toxin producer B. cereus.

    Quantification of Bacillus cereus stress responses
    Besten, H.M.W. den - \ 2010
    Wageningen University. Promotor(en): Marcel Zwietering; Tjakko Abee, co-promotor(en): Roy Moezelaar. - [S.l. : S.n. - ISBN 9789085857143 - 216
    bacillus cereus - stressreactie - zoutgehalte - zouttolerantie - hittetolerantie - adaptatie - weerstand - stresstolerantie - bacillus cereus - stress response - salinity - salt tolerance - heat tolerance - adaptation - resistance - stress tolerance
    The microbial stability and safety of minimally processed foods is controlled by a deliberate combination of preservation hurdles. However, this preservation strategy is challenged by the ability of spoilage bacteria and food-borne pathogens to adapt to stressing environments providing cell robustness. Bacillus cereus is a toxin-producing, spore-forming bacterium, and is able to survive minimal processing conditions. A quantitative approach was followed to gain insight in B. cereus’ stress adaptive behavior at population, individual cell and molecular level.
    B. cereus’ ability to adapt to salt stress and gain robustness towards subsequent heat challenge-stress exposure was quantified in detail using primary kinetics models. The adaptive salt stress response was influenced by the adaptation-stress concentration, the growth phase of the cells, strain diversity and the culturing temperature during adaptation-stress treatment. The nonlinear nature of the heat inactivation kinetics suggested heterogeneity within the population with respect to stress adaptive behavior. The direct-imaging-based Anopore technology was used to quantitatively describe the population heterogeneity of B. cereus upon mild and severe salt stress treatments and during low temperature growth. Fluorescent labeling of cells provided insights in the origin of stress-induced population heterogeneity. Then, to elucidate adaptive salt stress responses at molecular level, the genome-wide transcriptome profiles of mildly and severely salt-stressed cells were compared. Various transcriptome responses could be correlated to phenotypic features of salt stress-adapted cells. Comparison of the transcriptome profiles of salt stress-adapted cells to those that were exposed to mild heat, acid and oxidative stress, directed to potential cellular biomarkers for stress adaptation. The selected candidate-biomarkers  the transcriptional regulator B (activating general stress responses), catalases (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)  were measured upon adaptation-stress treatment at transcript, protein and/or activity level, and their induction was correlated to adaptation-stress induced robustness towards challenge-stress. Various candidate-biomarkers were suitable to predict the robustness level of adaptation-stress pretreated cells towards challenge-stress, and are therefore potential predictive cellular indicators for adaptation-stress induced robustness. The predictive potential of transcripts differed from that of proteins and activity level, underlining the significance to evaluate predictive potential of candidate-biomarkers at different functional cell levels. This quantitative understanding of B. cereus’ stress adaptive behavior provides mechanistic insights and opens up avenues to come to a mechanism-based approach for designing mild preservation strategies.
    Bacillus cereus acid stress responses
    Mols, J.M. - \ 2009
    Wageningen University. Promotor(en): Tjakko Abee; Marcel Zwietering, co-promotor(en): Roy Moezelaar. - [S.l. : S.n. - ISBN 9789085854944 - 176
    bacillus cereus - stressreactie - voedselbewaring - bacillus cereus - stress response - food preservation
    Bacillus cereus is a ubiquitous Gram-positive organism, which frequently causes foodborne
    illnesses. The widespread prevalence of B. cereus makes it a common contaminant in
    fresh foods where it also can cause spoilage. To prevent food-borne diseases and food
    spoilage, foods are often processed and/or preserved. In recent years, consumers’
    preferences have directed to fresher and tastier foods and this has acted as a driver for food
    industry to use milder processing and preservation techniques. Examples of hurdles that can
    be applied to preserve foods are low pH and the addition of organic acids. B. cereus may
    overcome these adverse conditions by displaying an adaptive stress response. The response
    of B. cereus upon exposure to these hurdles was investigated using two model strains,
    ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific
    genes and differences in metabolic capacities, including a urease encoding gene cluster in
    ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC
    10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e.,
    the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates.
    However, the activity appeared to be too low to contribute to acid resistance in the strains
    tested. To search for other acid resistance mechanisms, comparative phenotype and
    transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic
    and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the
    addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of
    the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed.
    Furthermore, a major oxidative response was displayed, which included the induction of
    several oxidative stress related genes and the production of inactivation-associated reactive
    oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were
    detected using fluorescent probes in combination with flow cytometry, including a newly
    developed method using a specific probe that enables superoxide detection in Grampositive
    and Gram-negative bacteria. The formation of ROS was also shown upon exposure
    to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus
    stress survival capacity revealed increased heat- and acid-resistance with cells grown and
    exposed to stresses in the absence of oxygen. The excess ROS may originate from stressinduced
    dysfunction of the aerobic electron transfer chain, which was indicated by the
    induction of alternative electron transfer chain components upon exposure to organic and
    inorganic acid shocks. Upon exposure to stress, superoxide is generated through the
    premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated
    rates. Subsequently, superoxide may promote the formation of other ROS, which can cause
    cellular damage leading to cell death. The induction of oxidative stress related genes has
    been reported in numerous other studies involving a wide range of bacteria exposed to
    different adverse conditions. However, a clear relation between the formation of ROS and
    the applied environmental stress was up to now not established. Secondary oxidative responses, including the formation of ROS, are possibly common bacterial responses to
    severe stresses under aerobic conditions. This thesis describes genomic differences between
    B. cereus strains and the acid stress response of these strains on transcriptome and
    phenotype levels, including measurements of intracellular ROS. The findings in this study
    can contribute to further understanding of bacterial stress responses and secondary
    oxidative responses. Furthermore, the results obtained may aid to optimize and select
    (combinations of) stresses to apply in hurdle technology, thus enabling design of safe,
    milder food processing and preservation techniques.
    Two-component signal transduction in Bacillus cereus and closely related bacteria
    Been, M.W.H.J. de - \ 2009
    Wageningen University. Promotor(en): Tjakko Abee; R.J. Siezen, co-promotor(en): C. Francke. - [S.l.] : S.n. - ISBN 9789085854357 - 176
    bacillus cereus - stressreactie - fylogenetica - signaaltransductie - genregulatie - bacillus cereus - stress response - phylogenetics - signal transduction - gene regulation
    Bacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a-typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments.

    Bacillus cereus associated food borne disease : quantitative aspects of exposure assessment and hazard characterization
    Wijnands, L.M. - \ 2008
    Wageningen University. Promotor(en): Tjakko Abee; Marcel Zwietering, co-promotor(en): Rijkelt Beumer. - [S.l. : S.n. - ISBN 9789085048848 - 176
    bacillus cereus - ziekten overgebracht door voedsel - blootstelling - risicoschatting - voedselhygiëne - blootstellingsbepaling - bacillus cereus - foodborne diseases - exposure - risk assessment - food hygiene - exposure assessment
    The consumption of food contaminated with the bacterium Bacillus cereus may lead to either symptoms of vomiting or symptoms of diarrhoea. As the symptoms are rather mild, few patients seek medical attention. Therefore, it is hard to estimate the number of cases. To improve estimation of this number and to gather more insight into factors that may determine the chance for disease, the following aspects have been investigated: the prevalence of B. cereus in food commodities in the Netherlands, the survival of B. cereus in simulated gastric fluid and the growth potential in simulated intestinal fluid, the interaction between B. cereus and intestinal cells, and the production of toxins that cause the disease symptoms. Together with literature data the results of these investigations have been used to determine the chance for disease by B. cereus. In combination with the number of meals that are consumed and that are contaminated with B. cereus, the yearly number of disease cases in the Netherlands has been estimated using different scenarios.
    Fine tuning of the Bacillus cereus stress respons: role of transcriptional regulators
    Voort, M. van der - \ 2008
    Wageningen University. Promotor(en): Tjakko Abee; Willem de Vos. - [S.l.] : S.n. - ISBN 9789085048985 - 143
    bacillus cereus - stressreactie - microbiële fysiologie - genexpressie - transcriptie - transcriptiefactoren - regulator-genen - bacillus cereus - stress response - microbial physiology - gene expression - transcription - transcription factors - regulatory genes
    The bacterium Bacillus cereus is able to survive and grow in a high diversity of environments, including foods, such as milk and pasta. Consequently, B. cereus can cause foodborne illnesses and food spoilage. During its time in food B. cereus encounters many changes in its environment, as the food is for instance heated and eaten. In order to cope with these changes in its environment B. cereus is able to switch on different sets of genes. This so-called gene regulation is regulated by an array of regulators. The performed research expands the understanding of the role of these regulators in fine tuning of gene regulation in response to changing environment in food.
    Germination of Bacillus cereus spores : the role of germination receptors
    Hornstra, L.M. - \ 2007
    Wageningen University. Promotor(en): Tjakko Abee; Willem de Vos. - [S.l.] : S.n. - ISBN 9789085046721 - 154
    bacillus cereus - bacteriële sporen - sporenkieming - voedselveiligheid - bacillus cereus - bacterial spores - spore germination - food safety
    The Bacillus cereus sensu lato group forms a highly homogeneous subdivision of the genus Bacillus and comprises several species that are relevant for humans. Notorious is Bacillus anthracis, the cause of the often-lethal disease anthrax, while the insect pathogen Bacillus thuringiensis is of economical importance, being used worldwide as an insecticide. B. cereus is a food pathogen, and can cause two types of foodborne diseases leading to an emetic- or diarrheal syndrome. The capacity of these bacteria to form highly resistant dormant particles, called spores, enables them to survive extreme conditions. Under favorable conditions, spores may germinate, herewith instantaneously loosing their resistance capacities, and grow out to vegetative cells. The research in this thesis describes the germination of spores of B. cereus. Precise timing of germination is crucial for survival. Therefore, the spore contains specific sensors, called germination receptors, which monitor the environment continuously for components necessary for growth. By systematically disrupting the complete set of B. cereus ger operons, coding for germination receptors, we revealed new germination-initiating molecules for the receptors. The GerR receptor was found to have a prominent role in germination, and appeared to be involved in initiation of germination by most of the amino acid- and purine ribosides. GerG showed participation in L-glutamine-initiated germination, while GerI played a role in purine riboside-initiated germination and the combined amino acid/purine riboside responses. Exposure of B. cereus spores to Caco-2 cells, that mimic the epithelial layer of the human small intestine, induced a strong germination response. The involvement of the germination receptors in this response was shown and found to be mainly mediated by GerI. It was furthermore shown that the nutrient content during sporulation defined the transcription levels of the ger operons and affected spore germination properties, suggesting that sporulation conditions influence the number of Ger receptors in the spore. The obtained knowledge was used to develop an improved cleaning-in-place method for inactivation of attached B. cereus spores. These spores are a frequent problem in processing equipment used in the food industry and the cause of a continuous source of contamination. The results presented are expected to contribute to a better understanding of Bacillus spore germination, and to facilitate the development of new food preservation strategies that will contribute to a better control of spores in our food products.
    Modeling to control spores in raw milk
    Vissers, M. - \ 2007
    Wageningen University. Promotor(en): J.M.G. Lankveld, co-promotor(en): M.C. te Giffel; P. de Jong. - [S.l.] : S.n. - ISBN 9789085046738 - 143
    rauwe melk - bacteriële sporen - boterzuurbacteriën - bacillus cereus - voedselbesmetting - microbiële besmetting - melkhygiëne - zuivelhygiëne - simulatiemodellen - raw milk - bacterial spores - butyric acid bacteria - bacillus cereus - food contamination - microbial contamination - milk hygiene - dairy hygiene - simulation models
    A modeling approach was used to identify measures at the farm that reduce transmission of microorganisms to raw milk. Butyric acid bacteria (BAB) and Bacillus cereus were used as case-studies. Minimizing the concentration of BAB spores in raw milk is important to prevent late-blowing of Gouda-type cheeses. Reducing the concentration of B. cereus spores in raw milk increases the shelf life of refrigerated pasteurized dairy products.

    First, predictive models were developed based on a translation of contamination pathways into chains of unit-operations. Via simulations, strategies were identified to control spore concentrations in farm tank milk (FTM)below 1,000 spores/L.Subsequently, the identified strategies were validated using data from a year-long field survey held at 24 Dutch farms.

    The results of this study show that mathematical modeling is very useful to identify effective measures to reduce the contamination of FTM with spores. The control strategies derived using model simulations were in agreement with results from the field survey. The following general conclusions were drawn:

    ·       To minimize the concentration of BAB spores in FTM, it is by far most important to prevent growth of BAB in grass- and corn-silage. Farmers should aim for a concentration in grass- and corn-silage fed to cowsbelow 1,000 spores/g. To achieve this, it is essential to prevent oxygen penetration into the silage silo and to remove molded and deteriorated silage from the ration fed to the cows. Measures aimed at other parts of the contamination pathway, such as teat cleaning prior to milking, are much less effective.

    ·       The concentration of B. cereus spores in FTM isnormally below 1,000 spores/L.During housing and pasturing spores of B. cereus in FTM originate from feeds. Two critical factors could lead to concentrations above 1,000 spores/L. Firstly, the contamination teats with soil is a high risk because soil can contain high concentrations of B. cereus spores. Secondly, build-up of B. cereus in improperly cleaned milking equipment could lead to high spore concentrations in FTM.

    ·       Implementation of the measures identified in this study could make late-blowing of Gouda-type cheeses a rare incident and prolong the shelf life of refrigerated pasteurized consumer milk by approximately 10%.


    A modeling approach was used to identify measures at the farm that reduce transmission of microorganisms to raw milk. Spores of butyric acid bacteria (BAB) and Bacillus cereus were used as case-studies because of their relevance for the Dutch dairy industry..The following general conclusions were drawn:

    ·       To minimize the concentration of BAB spores in FTM, it is by far most important to prevent growth of BAB in grass- and corn-silage. Farmers should aim for a concentration in grass- and corn-silage fed to cowsbelow 1,000 spores/g. To achieve this, it is essential to prevent oxygen penetration into the silage silo and to remove molded and deteriorated silage from the ration fed to the cows. Measures aimed at other parts of the contamination pathway, such as teat cleaning prior to milking, are much less effective.

    ·       The concentration of B. cereus spores in FTM isnormally below 1,000 spores/L.During housing and pasturing spores of B. cereus in FTM originate from feeds. Two critical factors could lead to concentrations above 1,000 spores/L. Firstly, the contamination teats with soil is a high risk because soil can contain high concentrations of B. cereus spores. Secondly, build-up of B. cereus in improperly cleaned milking equipment could lead to high spore concentrations in FTM.

    ·       Implementation of the measures identified in this study could make late-blowing of Gouda-type cheeses a rare incident and prolong the shelf life of refrigerated pasteurized consumer milk by approximately 10%.

    Bacillus cereus spore formation, structure and germination
    Vries, Y.P. de - \ 2006
    Wageningen University. Promotor(en): Tjakko Abee; Willem de Vos; Marcel Zwietering. - [S.l. ] : S.n. - ISBN 9789085043690 - 127
    bacillus cereus - bacteriële sporen - sporenkieming - sporulatie - structuur - bacillus cereus - bacterial spores - spore germination - sporulation - structure
    Bacterial spores arespecializeddifferentiated celltypes fortypes,specificallydesigned for thesurvival of adverse conditions. Their structure is highly unique and very different from the structure of normal vegetative bacterial cells. Spores cause massive problems in the food industry, because their remarkable resistance allows them to survive food processing and conservation methods. The spore-forming Bacillus cereus is an important food-borne pathogen, is famousfor its ability to causefood poisoning, andisan importantspoilage organism inpasteurizeddairy products. The work presented in this thesis has focused on the formation, structure and germination of B. cereus spores. An easy and efficient way of producing synchronized and homogeneous B. cereus spore batches was developed, using a chemically defined medium in combination with an airlift fermenter system. This setup allowed precise monitoring and manipulation of key growth- and sporulation parameters. The conditions employed resulted in synchronous growth and sporulation, which facilitated gene-expression studies. The kinetics of expression of sigA , sigB , sigF and sigG followed the model developed for Bacillus subtilis , underscoring the conservation of sporulation mechanisms among bacilli. B. cereus was able to form spores on the chemically defined medium without glucose but with lactate as a main carbon source. Sporulation was not induced by nutrient limitation, because significant amounts of carbon and nitrogen sources were still present when the cells started to sporulate. The presence of glutamate delayed the final stages of sporulation, but not the moment of sporulation initiation. Clearly, the concentration of glutamate influenced key spore properties such as heat resistance and germination. The alternative sigma factor σ B , encoded by the sigB gene, is an important stress response regulator of B. cereus . An increase in sigB transcription was observed upon glucose depletion, coinciding with the transition from exponential growth to the stationary phase. This increase was specifically associated with the depletion of glucose. Deletion of sigB had a significant impact on spore heat resistance and spore germination properties. Spore heat resistance is caused by the physicochemical structure of the spore, which protects vital spore components such as membranes, proteins, and the DNA. A spin-probe-based Electron Spin Resonance (ESR) method for measuring the internal structure of intact bacterial spores was developed and applied, and provided the first direct data on the aqueous environment in the various compartments of B. subtilis and B. cereus spores. From the results obtained, it was concluded that the core cytoplasm is not in a glassy state. Instead, a three-dimensional molecular matrix incorporating free but highly viscous water exists in the core. Notably, neither heat activation nor partial germination (the excretion of DPA but not full rehydration and enzyme activity) altered the structural properties of the core matrix significantly. Complete germination resulted in the disappearance of the structure in the core, and a decrease of the micro viscosity in the core cytoplasm to levels encountered in normal vegetative cells. For a quantitative analysis of the behavior of individual spores in a large, germinating spore-population, a flow cytometry (FCM) method was developed and applied. By using several different fluorescent dyes, distinct germination parameters such as DNA accessibility and esterase activity were quantified. Finally, spore properties from a large number of B. cereus strains, including the B. cereus laboratory model strain ATCC14579 and a number of recent isolates from environmental and industrial settings were analyzed. The strains tested showed a large variation in heat resistance, and the majority had a higher heat resistance than the laboratory model strain. With respect to germination, many of the strains were less sensitive to the nutrients tested as compared to the laboratory model strain. Heat activation and ageing enhanced germination in response to several nutrients in various isolates. The knowledge that was gained and the methods that were developed in this study are expected to contribute to progress in the spore research field, and to enhanced spore control in the food industry.
    The alternative sigma factor sigmaB and the stress response of Bacillus cereus
    Schaik, W. van - \ 2005
    Wageningen University. Promotor(en): Tjakko Abee; Willem de Vos; Marcel Zwietering. - Wageningen : s.n. - ISBN 9789085041849 - 165
    bacillus cereus - stressreactie - pathogenen - voedselveiligheid - overleving - rna-polymerase - bacillus cereus - stress response - pathogens - food safety - survival - rna polymerase - cum laude
    cum laude graduation (with distinction) The bacterium Bacillus cereus is responsible for a large number of cases of foodborne illness across the world. It is also an important cause of spoilage of food, in particular of milk and dairy-products. The growth and survival of B. cereus in food or during an infection is for a large part determined by to what extent the bacterium can adapt to changes in its environment. This process is known as the stress response and the protein SigmaB has an important role in it. Stress in B. cereus leads to the rapid activation of SigmaB. Subsequently, SigmaB coordinates the transcription of a set of genes, which leads to an increased resistance to stress. For example, SigmaB contributes to the growth and survival of B. cereus at low and high temperatures. It also has a role in cellular metabolism, which may indirectly also contribute to stress resistance. The knowledge obtained on the role of SigmaB in the stress response of B. cereus may contribute to the development of new, efficient, and safe methods for the production of food.
    Improved applicability of nisin in novel combinations with other food preservation factors
    Pol, I.E. - \ 2001
    Wageningen University. Promotor(en): F.M. Rombouts; E.J. Smid. - S.l. : S.n. - ISBN 9789058083821 - 95
    voedselbewaring - nisine - listeria monocytogenes - bacillus cereus - food preservation - nisin - listeria monocytogenes - bacillus cereus

    General discussion

    Modern consumers nowadays, have a preference for more natural, mildly preserved food products with a fresh appearance over traditionally preserved products. Mild preservation techniques applied singly are usually not sufficient to control microbial outgrowth and combinations of measures are needed to ensure complete safe products (16). Bacteriocins, produced by lactic acid bacteria have been successfully used as biopreservatives in a number of food products to inhibit the growth of pathogenic and spoilage organisms (27). Up till now, nisin is the only bacteriocin that has been approved by the WHO to be used as a food preservative. Due to its restricted inhibition spectrum and the decreased solubility and heat sensitivity at neutral pH, application is still limited (10). The study described in this thesis aimed to increase the practical application of nisin by combinations with other biopreservatives or mild preservation techniques.

    Nisin and essential oils

    Essential oils, derived from plants, are known for their flavor characteristics. Many of the compounds found in essential oils possess antimicrobial activity (4, 9, 14, 22), and therefore are suitable candidates for mild food preservation in combination with nisin. The essential oils dramatically enhance the bactericidal activity of nisin at concentrations, which alone do not affect the bacterial cell counts of the foodborne pathogens Listeria monocytogenes and Bacillus cereus (chapter 2). Adaptation of these cells to lower temperatures resulted in an increased sensitivity towards nisin, possibly due to an altered membrane composition leading to a change in membrane fluidity or to an increased electrostatic interaction of nisin with phospholipids in the membrane caused by an increase in negative charges (8, 18 - 21, 31). Alternatively a decrease in lipid II content as a result of changes in the membrane composition might explain the decreased activity of nisin (5). Lowering the temperature had a negative influence on the synergy between nisin and the essential oils, which might result from the lower sensitivity of the cells towards essential oils at lower temperatures (28).

    The exact mechanism underlying this synergy is not exactly known. Both nisin and carvacrol cause a dissipation of the proton motive force as well as depletion of the internal ATP pool (6, 12, 23, 26, 30, 32, chapter 3). In combination, carvacrol enhances the membrane potential dissipating effect of nisin, at concentrations which do not affect the viable count of B. cereus . Apparently cells are able to cope with low concentrations of nisin and carvacrol. When concentrations increase, cells are no longer able to compensate for loss of membrane integrity and a synergistic reduction of the pH gradient and depletion of the intracellular ATP pool were observed. The reduction in internal ATP is not proportional to the increase in external ATP and no additional increase in external ATP was observed upon simultaneous exposure to nisin and carvacrol. This observation excludes increased leakage of ATP as an explanation for the synergistic depletion of the intracellular ATP pool. Consequently, the underlying mechanism of the synergistic inactivation of B. cereus is most likely not the increased poreforming ability of nisin by carvacrol. Presumably, the rate of ATP hydrolysis is increased upon simultaneous addition of nisin and carvacrol or the internal ATP pool is exhausted in an attempt to reenergize the membrane (1, 23, 29). Alternatively, the disturbance of the membrane permeability by carvacrol and nisin might lead to impairment of membrane bound enzymes like ATPase, resulting in a decreased ATP synthesis (15, 26).

    Nisin and PEF treatment

    In addition to essential oils, Pulsed Electric Field treatment was also found to improve the antimicrobial action of nisin against B. cereus. Synergy was only found when PEF treatment was spread over a period of 10 minutes to match the relevant inactivation time scale of nisin's action. The additional stress imposed by PEF treatment possibly facilitates the incorporation of nisin into the cytoplasmic membrane resulting in more or larger pores or pores with a longer lifetime (chapter 4). Further reduction of the intensities of the treatments was achieved by adding carvacrol as a third hurdle to the combination of nisin and PEF treatment (chapter 5).

    The fact that synergy was found between the three treatments renders the combination very interesting for mild food preservation. However, extrapolation of the results from labscale experiments in buffer systems to food model matrices is usually difficult and the influence of food ingredients on the efficiency of preservation techniques are not fully understood. The efficiency of PEF treatment against vegetative cells of B. cereus is not affected by proteins in skimmed milk (20 %). However, the proteins do have a negative influence on the nisin activity, either as a result of a decreased bioavailability of nisin due to binding of the molecule to proteins or because of protection of the microorganisms by the proteins. As a consequence, the synergy between nisin and PEF treatment is less pronounced in skimmed milk (20 %).

    In sharp contrast to the improved bactericidal activity found in HEPES buffer, carvacrol is not able to enhance the synergy between nisin and PEF treatment in diluted milk (only in high concentrations (1.2 mM)). Possibly, carvacrol binds to the proteins, reducing the availability of the molecule. However, this is not consistent with the fact that carvacrol increases the antimicrobial activity of PEF treatment in milk. Therefore, the absence of synergy between nisin, PEF treatment and carvacrol is more likely explained by the decreased bioavailability of nisin, thereby decreasing the extent of synergy between nisin and carvacrol and consequently between all three treatments. The influence of PEF treatment on the behavior of proteins is not exactly known. Proteins can carry electric charges and might behave as dipoles when subjected to PEF treatment, which cause the macromolecules to reorient or deform (such as protein unfolding and denaturation), and possibly some breakdown of covalent bonds or casein micelles may occur (3). These PEF induced changes in the structure of proteins may play a role in the existence of synergy between carvacrol and PEF. Dilution of the milk to 5 % still provides enough proteins to stimulate synergy between carvacrol and PEF treatment (chapter 5).

    Before such novel techniques can replace currently used thermal processes, more insight into spore inactivation is needed (chapter 6). Nisin and PEF treatment do not directly inactivate or damage spores of B. cereus , however germinated spores can be inactivated by nisin or PEF treatment to a certain extent. The PEF resistance of the germinated spores is lost 50 minutes after the onset of germination. Nisin resistance was lost immediately in parallel to heat resistance, suggesting that loss of nisin resistance might be ascribed to changes in the dehydrated state of the core. Sulfhydryl groups in the membrane, not available in ungerminated spores, were suggested to be the natural target for nisin and therefore access to the membrane is a prerequisite for inactivation (17, 24, 25). In addition, the increase in availability of the membrane-anchored cell wall precursor Lipid II upon germination could also play a role in the loss of nisin resistance (5). Apparently, nisin has gained access to the membrane by penetrating the coat, which was made more permeable upon germination or alternatively, the protective coat was degraded by spore lytic enzymes, allowing nisin to reach the cytoplasmic membrane. The late loss of PEF resistance can be explained by its dependence on the degradation of the spore coat. To exert antimicrobial inactivation by PEF treatment, free migration of ions is needed to increase the transmembrane potential of the spores. Formation of pores occurs after compression of the membrane and reorientation of the phospholipids in the membrane. In spores the ions are immobilized by proteins or DPA, restricting their mobility (7, 13) and subsequently the build up of an increased transmembrane potential is prevented. Secondly, the spore core is surrounded by several rigid protecting layers limiting the compression and reorientation of the phospholipids (2).

    Combining nisin and PEF treatment did not result in additional inactivation of the germinating spores. Since loss of PEF resistance occurs only after 50 minutes of germination and loss of nisin resistance seems to be an early event in spore germination, synergy would therefore be less likely due to different time scales of action. Furthermore, the incomplete germination of the spores reduces the margins to observe synergy. Ideally, complete and synchronized germination is needed to quantify the inactivation by nisin or PEF treatment and determine precisely the onset of loss of nisin or PEF resistance.

    One of the main problems associated with the use of antimicrobial compounds is the development of tolerance or resistance to certain compounds. Adaptation of cells to carvacrol was correlated to a decrease in membrane fluidity as demonstrated by Ultee et al. (30). In addition, they observed a change in phospholipid composition of the membrane. Cells adapted to carvacrol exhibited an increased sensitivity towards nisin compared to control cells (chapter 6). A decrease in the membrane fluidity is not expected to increase nisin's action, but a change in the head group composition, with an increase in negatively charged lipids, might stimulate the electrostatic binding of nisin and in this way enhance nisin's action (8, 18 - 21, 31). Alternatively an increase in lipid II content in carvacrol-adapted cells as a result of changes in the membrane composition might explain the increased activity of nisin (5). A decrease in the membrane fluidity did not change the susceptibility towards a PEF treatment. A more rigid membrane is less likely to be compressed by accumulating charges as a result of applied field strength and the ordered state of the phospholipids in the membrane decreases the chance of reorientation, which would reasonably lead to a decreased inactivation by PEF treatment. Although the bactericidal activity of nisin was increased by adaptation to carvacrol, the synergy between nisin and PEF treatment was not influenced by a change in membrane fluidity and membrane composition. Attemps to change the membrane composition of spores by adaptation of vegetative cells to carvacrol prior to and during sporulation did not lead to inactivation of spores by either nisin or PEF treatment.

    Application

    Combinations of nisin with essential oils or PEF treatment have been successful in overcoming the restrictions in practical application of nisin. For instance, the inhibition spectrum of nisin can be widened by combination with other preservation technologies like PEF treatment. In addition, the limited activity of nisin at higher temperatures can be complemented by the increased synergy between nisin and essential oils.

    The application of multiple hurdles has great potential to be used as a mild food preservation technology. The occurrence of synergy between nisin and essential oils or PEF technology allows for a reduction in the intensities of the treatments demonstrating the suitability for mild preservation. Increasing the number of hurdles (lysozyme) improves the observed synergy and further increases the mildness of the preservation technology (chapter 1).

    Consumer's acceptation of these combination techniques in case of the essential oils is not expected to meet difficulties. This combination meets with present preference for more natural and mild preservation methods. Herbs and spices, of which essential oils are the active components, are already used for centuries as flavoring agents and in homeopathic products and medicines. Currently, carvacrol is Generally Recognized As Safe (GRAS) and has been approved by the Code of Federal Regulation (CFR) to be used as a flavoring agent (11). However, when the essential oils are used for their antimicrobial activity, they will be regarded as new food additives and subsequently require a non-toxicity report (27). To circumvent these problems, the original herbs and spices can be used as food flavoring agents, while at the same time advantage can be taken of their antimicrobial activity. However, the producer has to take into account the low concentration of the active compound in herbs and spices. Furthermore, the essential oils have a strong and specific flavor and can only be applied in products where this aroma is appreciated.

    Acceptance of PEF technology is expected to give more problems and introduction of this technology has to be handled carefully. Consumers might associate PEF treated foods with residual electromagnetic raditation, just like radiated foods are associated with radioactivity. Only when PEF technology is introduced carefully and the consumers are supplied with the right information, they will accept this technology as mild preservation.

    At the moment, not enough information is known about PEF technology and its mechanism of action. Evidently, more research needs to be done to verify the influence of other food ingredients including fat particles on the antimicrobial activity. Furthermore the influence of PEF treatment on the product quality needs to be investigated. The fresh-like appearance, color and the vitamin content are seemingly unaffected however, the influence of PEF treatment on proteins, polysaccharides macromolecules, or lipids is not exactly known.

    The development of tolerance or resistance to the PEF treatment or the combination treatments is not clear and should receive more attention, since microorganisms generally adapt to environmental stress factors. Increased tolerance towards nisin and carvacrol has been studied in more detail (8, 18 - 21, 31) however, no such research has been conducted concerning PEF technology. Combining preservation technologies in which the microorganism is attacked from different sides should reduce the development of tolerance to a minimum. Inactivation of spores is another challenge to be overcome before such combination technologies can be implemented in current preservation strategies.

    In conclusion, these combination techniques are a welcome alternative to currently used pasteurization methods. The current limitations in the application of nisin can be complemented by the inhibition spectrum of the combination treatment. In addition, the synergy observed between the different preservation techniques allows for a reduction of the used intensities increasing the suitability for mild preservation.

    References

    1. Abee, T., F. M. Rombouts, J. Hugenholtz, G. Guihard, and L. Letellier. 1994. Mode of action of Nisin Z against Listeria monocytogenes Scott A grown at high and low temperatures. Applied and Environmental Microbiology 60(6):1962-1968.
    2. Barbosa-Cánovas, G. V., M. Marcela Góngora-Nieto, U. R. Pothakamury, and G. S. Barry. 1999. Preservation of foods with pulsed electric fields. Academic Press, San Diego.
    3. Barsotti, L., P. Merle, and J. C. Cheftel. 1999. Food Processing by pulsed electric fields. I. Physical aspects. Food Review International 15(2):163-180.
    4. Beuchat, L. R., M. R. S. Clavero, and C. B. Jaquette. 1997. Effects of nisin and temperature on survival, growth and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy. Applied and Environmental Microbiology 63(5):1953-1958.
    5. Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H-G. Sahl, and B. de Kruijff. 1999. Use of the cell wall precursor lipid II by the pore-forming peptide antibiotic. Science 286:2361-2364.
    6. Bruno, M. E. C., A. Kaiser, and T. J. Montville. 1992. Depletion of proton motive force by nisin in Listeria monocytogenes cells. Applied and Environmental Microbiology 58(7):2255-2259.
    7. Carstensen, E. L., and R. E. Marquis. 1974. Dielectric and electrochemical properties of bacterial cells. In Spores VI. Michigan, 10-13 October 1974.
    8. Crandall, A. D., and T. J. Montville. 1998. Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype. Applied and Environmental Microbiology 64(1):231-237.
    9. Deans, S. G., and G. Ritchie. 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5 : 165-180.
    10. Delves-Broughton, J., and M. J. Gasson. 1994. Nisin, p. 99-131. In V. M. Dillon and R. G. Board (ed.), Natural antimicrobial systems and food preservation, vol. 328p. Cab international, Oxon, UK.
    11. Fenaroli, G. 1995. Fenaroli's handbook of flavor ingredients, third ed. CRC Press, Boca Rotan.
    12. Garcerá, M. J. G., M. G. L. Elferink, A. J. M. Driessen, and W. N. Konings. 1993. In vitro pore-forming activity of the lantibiotic nisin. Role of protonmotive force and lipid composition. European Journal of Biochemistry 212:417-422.
    13. Gould, G. W., and G. J. Dring. 1971. Biochemical mechanism of spore germination. In Spores V. Fontana, Wisconsin, 8-10 October.
    14. Kim, J. M., J. A. Marshall, J. A. Cornell, J. F. Preston III, and C. I. Wei. 1995. Antibacterial activity of carvacrol, citral and geraniol against Salmonella typhimurium in culture medium and on fish cubes. Journal of Food Science 60(6):1364-1368.
    15. Knobloch, K., H. Weigand, N. Weis, H-M. Schwarm, and H. Vigenschow. 1986. Action of terpenoids on energy metabolism, p. 429-445. In E. J. Brunke (ed.), Progress in essential oil research. de Gruyter, Berlin.
    16. Leistner, L., and L. G. M. Gorris. 1995. Food preservation by hurdle technology. Trends in Food Science and Technology 6(2):41-46.
    17. Lui, W., and J. N. Hansen. 1990. Some chemical and physical properties of nisin, a small protein antibiotic produces by Lactococcus lactis. Applied and Environmental Microbiology 56(8):2551-2558.
    18. Mazzotta, A. S., and T. J. Montville. 1999. Characterization of fatty acid composition, spore germination and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Applied and Environmental Microbiology 65(2):659-664.
    19. Mazzotta, A. S., A. D. Crandall, and T. J. Montville. 1997. Nisin resistance in Clostridium botulinum spores and vegetative cells. Applied and Environmental Microbiology 63(7):2654-2659.
    20. Ming, X., and M. A. Daeschel. 1993. Nisin resistance of foodborne bacteria and the specific resistance responses of Listeria monocytogenes Scott-A. Journal of Food Protection 56:944-948.
    21. Ming. X., and Daeschel, M. A. 1995. Correlation of cellular phospholipid content with nisin resistance of Listeria monocytogenes Scott A. Journal of Food Protection 58:416-420.
    22. Moleyar, V., and P. Narasimham. 1986. Antifungal activity of some essential oil components. Food Microbiology 3:331-336.
    23. Montville, T. J., and Y. Chen. 1998. Mechanistic action of pediocin and nisin: recent progress and unresolved questions. Applied Microbiology and Biotechnology 50:511-519.
    24. Morris, S. L., and J. N. Hansen. 1981. Inhibition of Bacillus cereus spore outgrowth by covalent modification of a sulfhydryl group by nitrosothiol and iodoacetate. Journal of Bacteriology 148(2):465-471.
    25. Morris, S. L., R.C. Walsh, and J. N. Hansen. 1984. Identification and characterization of some bacterial membrane sulfhydryl groups which are target of bacteriostatic and antibiotic action. The Journal of Biological Chemistry 259(21) : 13590-13591.
    26. Okereke, A., and T. J. Montville. 1992. Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679. Applied and Environmental Microbiology 58(8):2463-2467.
    27. Smid, E. J., and L. G. M. Gorris. 1999. Natural antimicrobials for food preservation, p. 285-308. In M. Shafiurr Rahman (ed.), Handbook of food preservation. Marcel Dekker, Inc., New York.
    28. Ultee, A., L. M. G. Gorris, and E. J. Smid. 1998. Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus . Journal of Applied Microbiology 85:211-218.
    29. Ultee, A., E. P. W. Kets, and E. J. Smid. 1999. Mechanisms of action of carvacrol on the foodborne pathogen Bacillus cereus . Applied and Environmental Microbiology 65:4606-4610.
    30. Ultee, A., E. P. W. Kets, M. Alberda, F. A. Hoekstra, and E. J. Smid. 2000. Adaptation of the foodborne pathogen Bacillus cereus to carvacrol. Archives of microbiology 174:233-238.
    31. Verheul, A., N. Russel, J., R. Van 't Hof, F. M. Rombouts, and T. Abee. 1997. Modifications of membrane phospholipid composition in nisin-resistant Listeria monocytogenes Scott A. Applied and Environmental Microbiology 63(9):3451-3457.
    32. Winkowski, K., M. E. C. Bruno, and T. J. Montville. 1994. Correlation of bioenergetic parameters with cell death in Listeria monocytogenes cells exposed to nisin. Applied and Environmental Microbiology 60:4186-4188.
    Carvacol krachtig natuurlijk conserveermiddel : trends in ingredienten : dl. 12
    Ultee, A. ; Smid, E. - \ 2000
    Voedingsmiddelentechnologie 33 (2000)12. - ISSN 0042-7934 - p. 11 - 14.
    voedselbewaring - voedselvergiftiging - anti-infectieuze middelen - bacillus cereus - voedingsmiddelen - smakelijkheid - aromatische stoffen - plantaardige oliën - secundaire metabolieten - voedingsmiddelenwetgeving - wetgeving - houdbaarheid (kwaliteit) - opslag - bacteriële toxinen - toxinen - food preservation - food poisoning - antiinfective agents - bacillus cereus - foods - palatability - flavourings - plant oils - secondary metabolites - food legislation - legislation - keeping quality - storage - bacterial toxins - toxins
    Deze natuurlijke stof fungeert als antioxidant, aroma en als conserveermiddel. Als conserveermiddel is Carvacrol niet toegestaan, wel als smaakstof
    Stress-respons van voedselpathogenen bij milde conservering
    Wouters, J.A. ; Bennik, M.H.J. ; Abee, T. - \ 2000
    Voedingsmiddelentechnologie 33 (2000)26. - ISSN 0042-7934 - p. 11 - 14.
    voedselbewaring thuis - voedselbewaring - houdbaarheid (kwaliteit) - kwaliteit - pathogenen - voedselmicrobiologie - voedingsmiddelen - genoomanalyse - genomen - listeria monocytogenes - bacillus cereus - voedselbederf - home food preservation - food preservation - keeping quality - quality - pathogens - food microbiology - foods - genome analysis - genomes - listeria monocytogenes - bacillus cereus - food spoilage
    Milde proces- en conserveringsmethoden en het karakteriseren van bederfveroorzakende micro-organismen op bepaalde behandelingen. De genoomsequenties van genoemde pathogenen werden onderzocht in relatie tot hoge osmolariteit, lage temperatuur en ultra hoge druk (UHD)
    Bactericidal action of carvacrol towards the food pathogen Bacillus cereus : a case study of a novel approach to mild food preservation
    Ultee, A. - \ 2000
    Agricultural University. Promotor(en): F.M. Rombouts; E.J. Smid. - S.l. : S.n. - ISBN 9789058082190 - 96
    voedselvergiftiging - bacillus cereus - voedselbewaring - anti-infectieuze middelen - secundaire metabolieten - etherische oliën - food poisoning - bacillus cereus - food preservation - antiinfective agents - secondary metabolites - essential oils

    A new trend in food preservation is the use of mild preservation systems, instead of more severe techniques such as heating, freezing or addition of chemical preservatives. Carvacrol, a phenolic compound present in the essential oil fraction of oreganum and thyme, is known for its antimicrobial activity since ancient times. This thesis describes a study of the antimicrobial activity of carvacrol towards the foodborne pathogen B. cereus . Carvacrol shows a dose-related inhibition of growth of B. cereus . Concentrations of 0.75 mM and higher inhibit growth completely at 8°C. Below 0.75 mM, carvacrol extends the lag-phase and reduces the specific growth rate as well as the final population density. Exposure to 0.75-3 mM carvacrol decreases the number of viable cells of B. cereus exponentially. Spores are approximately two fold more resistant towards carvacrol than vegetative cells.

    The incubation and exposure temperature have a significant influence on the sensitivity of B. cereus to carvacrol. An increase of the growth temperature from 8°C to 30°C decreases the fluidity of the membrane of vegetative cells and as a consequence, B. cereus becomes less sensitive to carvacrol. The change in membrane fluidity is probably the result of a higher percentage of lower melting lipids in the membranes at 8°C (chemical process) as an adaptation to lower temperature. Cells need to maintain an adequate proportion of the liquid-crystalline lipid in the membrane, as this is the ideal physical state of the membrane. On the other hand, an increase of the exposure temperature from 8 to 30°C, reduces the viability again. This can be explained by an increase of the membrane fluidity at a higher temperature as a result of melting of the lipids (physical process). At a higher membrane fluidity, relatively more carvacrol can dissolve in the membrane and the cells will be exposed to relatively higher concentrations than at a lower membrane fluidity.

    Not only the temperature plays a role in the activity of carvacrol, also pH is an important factor. The sensitivity of B. cereus to carvacrol is reduced at pH 7, compared to other pH-values between pH 4.5 and 8.5.

    Carvacrol interacts with the cytoplasmic membrane by changing its permeability for cations such as K +and H +. Consequently, the dissipation of the membrane potential (Δψ) andΔpH leads to inhibition of essential processes in the cell, such as ATP synthesis, and finally to cell death. At carvacrol concentrations as low as 0.15 mM,Δψis completely dissipated, however the viable count of B. cereus is not affected.

    Vegetative cells of B. cereus can adapt to carvacrol when the compound is present at concentrations below the MIC-value. Compared to non-adapted cells, lower concentrations of carvacrol are needed to obtain the same reduction in viable count of adapted cells. Adapted cells were found to have a lower membrane fluidity, caused by a change in the fatty acid composition and head group composition of the phospholipids in the cytoplasmic membrane. Adaptation to 0.4 mM carvacrol increases the phase transition temperature of the lipid bilayer (T m ) from 20.5°C to 28.3°C. Addition of carvacrol to cell suspensions of adapted B. cereus cells decreases T m again to 19.5°C, approximately the same value as was found for non-adapted cells in the absence of carvacrol.

    Incubation of cooked rice in the presence of different carvacrol concentrations results in a dose-related reduction of the viable count of B. cereus . Concentrations of 0.15 mg/g and above, reduce the viable count, leading to full suppression of growth at 0.38 mg/g. The influence of carvacrol on the viable count is dependent on the initial inoculum size. Although carvacrol is an effective inhibitor of growth of B. cereus in rice, it could affect the flavour and taste of the product at concentrations where full suppression of growth is observed. However, strong synergistic activity is observed when carvacrol is combined with the biosynthetic precursor cymene or the flavour enhancer soya sauce. This makes it possible to use lower carvacrol concentrations and consequently a smaller influence on the sensoric properties of the rice is expected.

    Besides its influence on the viability of vegetative cells, carvacrol also shows inhibition of diarrhoeal toxin production by B. cereus at concentrations below the MIC-value. Addition of 0.06 mg/ml carvacrol to the growth medium, inhibits the toxin to 21% of the control (no carvacrol added). The inhibition correlates with the reduction of the viable count of B. cereus in the presence of carvacrol. At the same time, the total amount of cells did not change. In mushroom soup, also an inhibition of the toxin production was observed, however, the viable count did not change. This effect on the toxin production is most probably caused by a lack of sufficient metabolic energy, since carvacrol affects ATP synthesis. The cell will use its low levels of ATP to maintain its viability, rather than using it for toxin production or excretion. It could also be possible that the decreased toxin synthesis in BHI was the result of the lower amount of viable cells. The inhibition of toxin production at carvacrol concentrations which do permit growth of B. cereus , reduces the risk of food intoxication by this pathogen.

    In conclusion, carvacrol may play an important role in future as a natural antimicrobial compound. However, its application will most probably be in combination with other natural antimicrobial systems.

    Bacillus cereus in melk en stal
    Wolters, G. ; Slaghuis, B. ; Lely, L. van de - \ 1992
    Praktijkonderzoek / Praktijkonderzoek Rundvee, Schapen en Paarden (PR), Waiboerhoeve 5 (1992)6. - ISSN 0921-8874 - p. 16 - 18.
    huisvesting, dieren - dierlijke producten - kiemgetal - bacteriologie - hygiëne - melkkwaliteit - melkopbrengst - bacillus cereus - animal housing - animal products - bacterial count - bacteriology - hygiene - milk quality - milk yield - bacillus cereus
    Deze veehouders zullen moeten kunnen garanderen dat de melk, die zij voor directe verkoop aanbieden of die verwerkt wordt tot rauwmelkse kaas, vrij is van pathogenen (vrij van pathogenen in de betekenis van dusdanige lage aantallen dat er geen gevaar voor de volksgezondheid kan ontstaan).
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