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 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.

    Clostridium perfringens sporulation, germination and outgrowth in food: a functional genomics approach
    Xiao, Y. - \ 2014
    Wageningen University. Promotor(en): Tjakko Abee, co-promotor(en): M.H.J. Wells-Bennik. - Wageningen : Wageningen University - ISBN 9789462570160 - 138
    clostridium perfringens - bacteriële sporen - sporenkieming - sporulatie - functionele genomica - clostridium perfringens - bacterial spores - spore germination - sporulation - functional genomics

    At the heart of foodborne disease caused by Clostridium perfringenslays its ability to form spores. The ubiquitous presence of C. perfringensis due to the fact that spores are resilient and are able to survive harsh environmental conditions. As a result, spores of C. perfringenscan be found in many food ingredients and are able to survive a range of processing steps in the food industry. Subsequent spore germination in foods - followed by outgrowth - can lead to product spoilage and foodborne illness. Yet, not all C. perfringensstrains can cause foodborne illness; only certain C. perfringensstrains that produce enterotoxin (CPE) have this ability. Consumption of high levels (more than 105CFU/g) of such CPE producing strains can lead to diarrhea when cells sporulate, concomitant with release of the spores and CPE in the gut.

    One of the aims of this thesis was to obtain better insight in genes that play a role in the process of sporulation and germination of C. perfringenscompared with those in the better studied Bacillusspecies. This was achieved through in silicoanalysis of germination genes in the genomes of Clostridiumand Bacillusspecies. Overall, it was found that the numbers of gergenes, encoding germinant receptors of the GerA family, are lower in clostridia than in bacilli. Moreover, various Clostridiumspecies are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli.

    So far, studying gene function and regulation in clostridia has been hampered by the lack of genetic tools, but novel insights in genes putatively involved in sporulation and germination were obtained through whole genome transcriptome analysis during sporulation. The majority of previously characterized C. perfringensgermination genes showed significant upregulated expression profiles in time during sporulation. Such upregulated expression profiles during sporulation were also observed for other genes, including C. perfringenshomologs of Bacillussporulation and germination genes. A comprehensive homology search revealed that approximately half of the upregulated genes are conserved within a broad range of sporeforming Firmicutes; these genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior.

    Food borne disease by C. perfringenscan only be caused by strains that carry the cpegene, encoding the enterotoxin. This gene can be carried on the chromosome (C-cpe) or on a plasmid (P-cpe). To characterize C. perfringensstrains present in foods, isolates from a nationwide survey were subjected to 16S rDNA sequence analysis, multi locus sequence typing (MLST) and toxin gene profiling. This revealed that the current standard cultivation method gives false-positive results in ~30% of cases (i.e.species other than C. perfringenswere identified). Of the confirmed C. perfringensisolates, only ~10% carried the cpegene. For these cpe-positive strains, the gene was predominantly carried on the chromosome, but P-cpestrains were also found. MLST analysis showed that the C-cpestrains are evolutionarily distant from cpe-negative and P-cpestrains (with the latter two associated with the gut). Overall, these results highlight a need for improved methods to detect C. perfringenswith higher specificity, ideally simultaneously allowing for discrimination between C-cpe, P-cpeand cpe-negative strains.

    The growth potential of cpe-negative, C-cpeor P-cpe strains was further assessed in a model food. Spores were obtained from 15 diverse strains and spore heat resistance was the highest for C-cpestrains. Spores of the individual strains were inoculated in raw minced beef prior to vacuum packaging and heating, and their germination and growth potential was assessed during storage at 12°C and 25°C. This showed lower outgrowth potential of C-cpestrains than of cpe-negative and P-cpestrains at 12°C, suggesting that the cpe-negative and P-cpestrains may have a competitive advantage over C-cpestrains when these are present in products at low storage temperatures (refrigeration abuse temperatures). Overall, the C-cpestrains produce spores that are relatively heat resistant and if such spores survive (insufficient) heat treatments, they may be able to multiply rapidly if cooling regimes are not appropriate. Once the product reaches temperatures below 12°C, growth will be limited. Therefore, rapid cooling is very important and a critical control point. The P-cpestrains, on the other hand, produce spores that are rather heat sensitive and these will normally be inactivated even by relatively low heat treatments. However, if such strains are introduced as a post-processing contamination, for instance by food handlers, and the product is held for prolonged times at abuse refrigeration conditions, they may pose a risk for food borne infections. The post-processing hygienic measures in combination with adequate cooling may therefore be the most critical with respect to control of P-cpestrains.

    Overall, this thesis has provided new insights in the genes involved in germination of C. perfringensversus the well-known Bacillusspecies, and potential new candidate genes that play a role in sporulation and germination of spore forming Firmicutes were identified. It was apparent that cultivation methods that are currently available for C. perfringensrendered a large percentage of false positives. Further genetic analysis of C. perfringensisolates obtained from foods showed that C-cpestrains – which are usually associated with food - belong to a cluster that is distinct from P-cpeand cpe-negative strains. Strains belonging to the C-cpeand P-cpeclusters showed different heat resistance characteristics and outgrowth potential in a model food system at low temperatures. This points to different critical control points to prevent foodborne outbreaks due to C. perfringens; for C-cpestrains an adequate heat treatment is required to inactivate spores and rapid cooling is needed to prevent outgrowth in the event any spores survived. For P-cpestrains, spores are rather sensitive to heat treatment, but it is particularly important to prevent post-heating contamination and maintain low storage temperatures of products.

    Microbial ecology of the cocoa chain : quality aspects and insight into heat-resistant bacterial spores
    Líma, L.J.R. - \ 2012
    Wageningen University. Promotor(en): Marcel Zwietering, co-promotor(en): Rob Nout. - S.l. : s.n. - ISBN 9789461734334 - 311
    cacao - theobroma cacao - cacaoproducten - fermentatie - micro-organismen - bacteriële sporen - hitteresistentie - voedselveiligheid - cocoa - theobroma cacao - cocoa products - fermentation - microorganisms - bacterial spores - heat resistance - food safety

    Cocoa beans (Theobroma cacaoL.) are the basis for chocolate and cocoa powder production. The first step in the production of these food products consists of a spontaneous fermentation of the beans in the tropical producing countries, in order to allow the formation of the essential precursor compounds of the cocoa flavour. Following this stage, cocoa beans are industrially transformed into a range of different products.

    In the first part of this thesis, the state of literature of cocoa bean fermentation was revisited with the aim of interlinking post-harvest processing practices and characteristics of the fermenting microbiota, with the quality of commercial cocoa beans obtained. This literature survey resulted in the identification of a number of research needs that can be used to design agricultural and operational measures, towards improving the quality of commercial cocoa beans. The second part of this thesis addressed the occurrence, levels and diversity of bacterial Thermoresistant Spores (ThrS) in commercial cocoa powder and in cocoa being transformed to cocoa powder in an industrial setting. Thermoresistant Spores were defined as spores which survive a heat-treatment of at least 100°C for 10 min, as opposed to the standard treatment at 80°C for 10 min for Total Spores (TS) determination. It was found that strains of Bacillus licheniformis and of the B. subtilis complex were the predominant species in the ThrS library and that strains of B. subtilis complex, including B. subtilis subsp. subtilis formed the most heat-resistant spores in situ and in vitro. Interestingly, a high heat-resistant spore phenotype was associated with diverse genotypes. In addition, a strategy was devised in order to identify biomarkers of spore heat-resistance. The comparison of physicochemical and ultrastructural properties among spores of different heat-resistance allowed the identification of manganese as a potential physiological biomarker for B. subtilis subsp. subtilis spores heat-resistance. On the other hand, a study of various genomic features conducted across strains of the phylum of Firmicutes, did not yield genomic biomarkers related to spore high heat-resistant phenotype, suggesting the primary role of spore physicochemical composition and ultrastructural characteristics as determinant factors of spore differential heat-resistance.

    In conclusion, this study presents suggestions to delve into the functionality of the microbiota involved in cocoa bean fermentation and to improve the quality of commercial cocoa beans. Furthermore, it contributes towards the understanding of the ecology of high heat-resistant aerobic bacterial spores in the cocoa chain and provides knowledge regarding their heat-resistance. The data gathered in this thesis are useful input for predictive modelling and to assist in the production of safe and high quality 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.
    Sterilization by high hydrostatic pressure : increasing efficiency and product quality by improved temperature control
    Heij, W.B.C. de; Schepdael, L.J.M.M. van; Moezelaar, R. ; Berg, R.W. van den - \ 2003
    In: Advances in high pressure bioscience and biotechnology II : proceedings of the 2nd international conference on High pressure bioscience and biotechnology II / Winter, R., Berlin : Springer Verlag - ISBN 9783540009771 - p. 367 - 370.
    steriliseren - warmtebehandeling - bacteriële sporen - drukbehandeling - verwerkingskwaliteit - sterilizing - heat treatment - bacterial spores - pressure treatment - processing quality
    A product being pressurized will heat up due to compressive heating. Due to heat transfer, products close to the vessel wall will cool down, a process which may result in a non-homogeneous product temperature profile in radial direction. If the proper technological features are implemented these heat losses may be reduced significantly. Such an adiabatic-isobaric process increases the economic feasibility of the high pressure process and minimizes product damage due to a small temperature-time integral.
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