Stress-response and survival strategies of Bacillus cereus

Transcription

1 Stress-response and survival strategies of Bacillus cereus Bacillus cereus, sigma factors, polymerase, molecular biology, bioinformatics Marcel Tempelaars Bacillus cereus is a food pathogen that is often involved in outbreaks of food-borne illness. Because B. cereus is able to form spores, this organism can survive the production processes used in the food industry. These spores can germinate in the final food product and after that outgrowth might occur. Under these processing conditions the cells are exposed to several types of stress, such as a low or high temperature, high concentrations of salt, a lack of energy sources and/or a low ph. The central question in this research project is to elucidate how vegetative cells of B. cereus can withstand the different stresses they can encounter with a special emphasis on the role of regulators of gene expression such as sigma factors. Sigma factors are subunits of the enzyme RNA polymerase, which takes care of the transcription of DNA to RNA. Sigma factors recognize specific promoter elements and can therefore "switch on" specific sets of genes. The role of sigma factors in the stress-response of Bacillus cereus In this research project the role of different regulators of of the stress response of B. cereus will be characterized using wild type strains and selected deletion mutants. Using different techniques, such as mutant construction, phenotypic analysis, micro arrays, and by promoter-reporter fusions, the expression of genes and roles of corresponding enzymes/proteins will be determined. This molecular biological characterization can be coupled to the analysis of different physiological parameters (such as membrane integrity and energy status), with the aim to obtain insight in the signals that lead to the activation of adpative stress responses. Next, characterisation of the cellular defense systems involved and their impact on adaptation and survival can be determined.

2 Identification of genes and molecular mechanisms involved in biofilm formation Supervisors: Biofilm, pathogens, molecular biology Natalia Crespo (Listeria biofilms) Marcel Tempelaars (Bacillus biofilms) BSc and MSc 3-6 months Many bacteria are capable of colonizing surfaces and grow in structures called biofilms. Biofilms consist of the bacteria, attached to a surface and embedded in an extrapolymeric matrix, which can consist of polysaccharides, protein, DNA etc. Biofilm cells are relatively hard to remove, because they are more resistant to antimicrobial compounds and disinfectants. When food pathogens and spoilage organisms manage to establish biofilm growth in food processing equipment, they will form a persistent source of recontamination. The research in this project focuses on Gram-positive bacteria that can occur in a variety of food products such as Listeria monocytogenes and Bacillus cereus. Different stages of biofilm development To be able to prevent or fight biofilm formation, more knowledge is required on the genes and molecular mechanism behind this physiological process. The complete genome sequence of representatives of the bacteria listed above are available. The project concerns the identification of cellular parameters and metabolic pathways and signalling compounds involved in biofilm formation using wild type strains and deletion mutants. Differences in biofilm phenotypes will be determined and underlying mechanisms can be further analyzed using techniques like RT-PCR and RNAseq analysis.

3 Mixed biofilm formation of L. monocytogenes and lactic acid bacteria Supervisors: biofilms, Listeria monocytogenes, multi-species, molecular biology Natalia Crespo BSc and MSc 3-6 months Biofilm are surface associated structures of bacterial cells that are attached to a substratum, embedded in a matrix of extracellular polymeric substances. Bacteria in a biofilm have altered phenotypes compared to cells in liquids with respect to their growth rate and gene transcription, and in general more resistant to environmental adverse conditions. In natural environments, biofilms are composed of more than one species resulting in so-called mixed species biofilms. Listeria monocytogenes, the causative agent of listeriosis, can form biofilms and it is encountered in similar niches as biofilm forming L. plantarum. Therefore, they are good model organisms to study formation of mixed biofilms. To date, mechanisms involved in mixed species biofilm formation involving food-associated microbes including pathogens have hardly been studied. Therefore, we will study mixed species biofilm formation by L. plantarum in co-culture with L. monocytogenes model strains and targeted mutants. These L monocytogenes strains have deletions in specific genes, which might play a role in (mixed) biofilm formation. The impact of environmental conditions on mixed species biofilm formation will be analysed by different techniques including (fluorescence) microscopy analysis and counting of biofilm cells). Microscopic images of L. monocytogens and L. plantarum mixed biofilms

4 Spore resistance and germination capacity of mesophilic and thermophilic Bacillus species. Bacillus spp., sporulation, germination, growth performance Marcel Tempelaars Masja Nierop Groot (FBR) Spores produced by mesophilic and thermophilic sporeformers may be introduced and/or produced in food processing environments for example in so-called biofilms. In the latter case spores may be released into fluid streams in pipe lines and end up in the final product. Characterization of stress resistance and germination capacity of the spores produced in different conditions, may lead to development of more effective cleaning procedures and cleaning strategies. A combination of selected mesophilic and thermophilic microorganisms including respectively (enterotoxic and emetic) B. cereus and G. stearothermophilus isolated from foods and food processing environments will be included in the project. The following parameters will be investigated using a range of methods including: Spore formation capacity, spore stress resistance, germination capacity, and outgrowth performance using plate counts, (fluorescence) microscopy, and flow cytometry. References: - van der Voort M, Abee T. (2013) Sporulation environment of emetic toxin-producing Bacillus cereus strains determines spore size, heat resistance and germination capacity. J Appl Microbiol. 114(4): den Besten HM, van Melis CC, Sanders JW, Nierop Groot MN, Abee T. (2012) Impact of sorbic acid on germination and outgrowth heterogeneity of Bacillus cereus ATCC spores. Appl Environ Microbiol. 78(23):

5 Heterogeneity in germination and outgrowth of Bacillus cereus endospores B. cereus, sporulation, germination, outgrowth Marcel Tempelaars Masja Nierop groot (FBR) Bacillus cereus is a Gram-positive, spore-forming bacterium that can be found in nearly every environment. This means that it is easily transmitted into the food chain. The spores formed by B. cereus are highly resistant against stresses, and are thus able to survive several processing conditions used in the food industry. After germination in the final food product, outgrowth of B. cereus can occur, after which it is able to cause food spoilage or food poisoning due to production of toxins. In order to improve control of sporeformers, extensive research efforts are aimed at the improvement of preservation methods. This study focusses on the germination and outgrowth capacity of B. cereus spores with specific emphasis on the heterogeneity of these processes including the role of so-called superdormant spores that are not easily triggered by nutrient germinants. Previous research has shown that a large diversity occurs in germination between individual spores, even under similar conditions. While one spore is already germinated and growing, others will still be dormant. Heterogeneity in spore germination and outgrowth capacity will be studied at single cell level by flow cytometry (FCM) or by direct imaging on Anopore The impact of preservation treatments such as (mild) heating on spore germination and outgrowth capacity will be determined. An extension of the research may include the assessment of factors contributing to recovery of the (heat)damaged spores using selected deletion mutants. Planning of the project: 1. production of spores (different conditions) 2. stress exposure of spores 3. germination capacity (FCM) or outgrowth capacity (anapore method) 4. heterogeneity assessment Cells and spores in cells

6 Microbial warfare; understanding how small metabolites and peptides produced by one organism inhibit the growth of other microorganisms Antimicrobial compounds, Bacillus cereus, valinomycin, cereulide, flow cytometry Marcel Tempelaars Bacteria and fungi are known to produce substances that are secreted into their habitat and that may have antimicrobial properties against other microorganisms. These antimicrobial agents can be of importance for their potential as food preservatives and antagonistic effect against important pathogens. Similar compounds may also be produced by food-borne pathogens, such as the emetic toxin cereulide, that is produced by Bacillus cereus. This toxin is associated with the onset of the emetic syndrome, that is characterized by vomiting and nausea. Antimicrobial compounds are produced in a variety of environments and in many cases their relevance in survival and/or competitive advantage of the producing organism is not clear. In this project the effects of cereulide produced by B. cereus on intraspecies and interspecies interactions will be investigated. A range of techniques will be used including analysis of growth inhibition, viable counts, spore counts, live/dead analysis and cellular activity detection using fluorescence microscopy and flow cytometry. The results obtained in this project will contribute to our understanding of the impact of cereulide on ecology and performance in foods of emetic B. cereus.

7 Resistant variants in bacterial populations Supervisors: pathogens, stress resistance, characterization, kinetics Jeroen Koomen BSc and MSc 3-6 months Listeria monocytogenes is a food-borne pathogen causing the rare but severe disease listeriosis. Its robustness and ability to grow under harsh conditions make it a difficult pathogen to eliminate. An additional feature that can have implications for food safety is the occurrence of subpopulations of stress resistant variants of L. monocytogenes. The presence of such subpopulations can lead to tailing of the inactivation curves. This is a major concern for the food industry since tailing can lead to less inactivation than expected when only the linear part of the inactivation curve is taken into account in the design of safe preservation methods. Furthermore also more resistant strains might be selected for in industrial environments. This is especially a concern within the recent trend towards minimal processing. This trend is challenging food producers to design products that guarantee microbiologically safe product by using mild preservation techniques. The focus of this research is the occurrence of these stress resistant subpopulations. Exposure to low ph [1] results in inactivation curves exhibiting considerable tailing. A set of colonies isolated from the tail were confirmed to be more resistant than the wild type. The resistant isolates with a stable phenotype were further characterized for resistance to other stresses, growth and motility. Phenotyping of a set of resistant isolates revealed population diversity of L. monocytogenes LO28. The aim of this project is to further characterize the obtained set of stress resistant variants including bacteriophage resistance. We want to identify the mutations and corresponding phenotypes in the set of variants in order to get better insight in the mechanisms leading to increased stress resistance in L. monocytogenes. Additionally, it is of great interest to investigate L. monocytogenes strain diversity (soil, food, clinical isolates) and if the mutations leading to increased stress resistance are also found in other strains of L. monocytogenes. Acid inactivation kinetics of L. monocytogenes LO28 WT and acid resistant variants 1. K.I. Metselaar, H. M. W. Den Besten, T. Abee, R. Moezelaar, M.H. Zwietering (2013). "Isolation and quantification of highly acid resistant variants of Listeria monocytogenes." International Journal of Food Microbiology 166(3):

8 Inactivation of spores by cold plasma treatment Bacillus ssp., Bacillus cereus, mould spores, cold plasma, surface decontamination Masja Nierop Groot (FBR) Tjakko Abee (WU) The need for novel surface decontamination methods as alternative to currently applied heat and chemical based methods for food or food packaging material has directed focus towards non thermal plasma as candidate technology. Cold plasma targets spores, but effectiveness depends on plasma parameters such as design of electronic power circuits, carrier gas, but also characteristics of the microbe. Understanding of the antimicrobial mode of action of cold plasma will be a prerequisite for future regulatory status of non-thermal plasma application. Mechanisms involved in inactivation of spores by NCAP The objective of this project is to study the mechanism of nitrogen cold atmospheric plasma gas (NCAP) as low temperature, non-chemical based method for decontamination of bacterial or fungal spores on surfaces. This research project will focus on the mode of action of NCAP on spores. The project may involve Bacillus cereus or Bacillus subtilis wild type spores and those formed by selected deletion mutants to obtain insight in cellular parameters involved in survival. Spore stress resistance, germination capacity, and outgrowth performance using plate counts, (fluorescence) microscopy, and flow cytometry upon NCAP treatment will be studied. Moreover, the effect of NCAP treatment on mould spores could be a research topic. This will include an inventory of the effect of NCAP towards spores derived from different food relevant moulds.

9 Control of fungal spores in food products Supervisors: fungal spores, mild processing Masja Nierop Groot (FBR) Tjakko Abee (WU) MSc 6 months An increased demand for healthy and nutritious foods with increased freshness and shelf life and at the same extended shelf life poses new challenges in assuring the stability and safety of a variety of foods. Moulds can grow on cereals, fruits and fruit juices, beverages, dairy products, fermented products and consequently are associated with spoilage of a wide range of products. Not all products tolerate intensive heat treatments required to inactive mould spores as these compomise quality and nutritional aspects. Mild processing techniques in combination with secondary hurdles may be applied to realize stable products. In this research project, we will focus on the use of mild processing in combination with different matrix composition to control germination and outgrowht of yeast and mould spores. The project involved the use of different yeats species or spores produced by different moulds encountered in food. Mild techniques could involve the use of cold plasma, pulsed electric field (PEF) or high pressure processing in combination with variation in temperature, gas composition, antimicrobial compounds as conditions during storage.