Microbes and Function
Fungal food spoilage often starts with a contamination with spores. These reproductive structures are abundant in the environment. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation methods. The aim of this project is to study the extent of this heterogeneity and to study the underlying mechanisms using spores of Aspergillus niger, Paecilomyces variotii, Penicillium roqueforti, and Saccharomyces cerevisiae subsp. diastaticus as model systems. The role of the genetic background, environmental conditions, and the developmental state of the mycelium and spores will be studied. Quantitative imaging, genome and RNA sequencing, functional gene analysis, and modelling will be used, which should reveal leads for novel mild intervention protocols to prevent food spoilage.
- Study the impact of the genetic background by using a collection of strains of different geographic origin and originating from contaminated food and beverages.
- Study the impact of environmental growth conditions by isolating spores from colonies grown at different substrates either or not in the presence of sub-lethal stress conditions.
- Study the impact of the developmental stage of the colony and the conidia by isolating spores of different age from different zones of colonies.
- Show a proof of concept of a novel processing treatment that prevents fungal spoilage by making use of a combination of mild interventions.
- Description of the impact of the genetic background on variability in stress resistance between strains of the model fungi used in this project.
- Description of the impact of environmental growth conditions on variability in stress resistance between strains of the model fungi used in this project.
- Description of the impact of the developmental state of the mycelium and the spores on variability in stress resistance between strains of the model fungi used in this project.
- Molecules triggering germination of a (sub)population of spores of the model fungi used in this project.
- Mechanism(s) underlying spore heterogeneity with respect to stress resistance.
- Generic models describing growth/no growth boundaries and/or germination and outgrowth kinetics.
- Proof of concept of a novel processing treatment that prevents.
In engineering and economics trade-offs are well known. Similarly, evolutionary trade-offs in microbial cells are defined as the optimization of one trait at the cost of another. For instance if a cells puts lots of energy into the production of costly molecules like exo-polysaccharides little energy is available for cell growth.
This project focuses on the influence of trade-offs on industrial fermentations. We will investigate the role for key enzymes in dairy fermentations including enzymes involved in growth, (post-) acidification, flavor- and texture formation. Industrially relevant parameters will be investigated including temperature, salt, starvation and pH stress. These conditions change rapidly throughout cheese manufacturing and we will investigate how these changes influence functionality of the starter culture in the fermented dairy product. The results of this project are designed to allow us to develop new starter cultures, shorten lag-phases, increase flavor formation and shorten cheese ripening times and improve the robustness of the fermentation process.
- Proteome turnover in L. lactis under industrial relevant conditions.
- Enzymatic activity decay data on at least 10 flavor and growth related enzymes at industrially relevant conditions.
- Impact of environmental transitions on heterogeneity, outgrowth and fermentation times in milk.
- Modulation of the catalytic capacity of a starter culture.
- Extended metabolic model to identify trade-offs based on data generated in the deliverables above.
- Alteration of starter functionalities (in whey, milk and cheese) by exploiting trade-offs.
The detection and characterisation of microbial contaminants in raw materials, intermediate and end products is important for improving product quality and process efficiency. The Detection project has addressed research needs that include concentration methods for the detection of extremely low numbers of microorganisms (Concentration), insight into the composition and outgrowth of the microbial spoilage population (Composition) and insight into the physiological state of spoilage microorganisms in order to reduce their survival in food processing and preservation conditions (Physiological state).
In the Concentration sub-project we developed milk pre-treatment methods based on proteolytic digestion. These methods have allowed the entrapment and enumeration of microorganisms on a microsieve. Experiments for the detection of spores in clear media by the microsieve method showed a detection limit of approximately 103 spores per ml. In the Composition sub-project we applied mass sequencing to monitor the spoilage population in a ready-to-eat rice meal, during spontaneous spoilage in the absence or presence of weak organic acids, in order to assess the effects of these acids on outgrowth and compare the results to those obtained by conventional plate counting. In addition, we monitored the presence and outgrowth of a mixture of bacterial spores in a soup matrix using a very similar approach. In the Physiological state sub-project, analysis of heterogeneity in the heat-stress-resistance of Bacillus subtilis was carried out using live/dead stains followed by single-cell analysis. The differences observed in stress sensitivity appeared subtle.
The project was successfully completed in 2013 and has resulted in a paper in the peer-reviewed journal Applied Environmental Microbiology (Yu et al), and a cross-project collaboration with Biofilm and Detection – another project within the theme Food Safety and Preservation. The project also delivered a manuscript with active input from the two industry partners involved in the project. Moreover, the outcomes were received with interest at the SPOILERS 2013 congress in Quimper (France). A review on cultivation-independent detection of extremely low amounts of micro-organisms is under preparation, initiated by Wageningen UR Food & Biobased Research – one of the research partners in the project.
Milk fermentation is a key process in adding functional properties, such as flavour, texture and shelf life, to dairy products. Cheese starter-cultures consist of simple or complex mixtures of Lactococcus lactis strains, sometimes together with other species of the genera Leuconostoc and/or Lactobacillus. These bacteria actively cooperate in microbial consortia.
We have learned, during the last decade, that not all cells within a pure bacterial culture exhibit the same behaviour. It is reasonable to assume that such culture heterogeneity also applies to industrial fermentations. We use Lactococcus lactis as a model organism for dairy fermentation and, by using fluorescent proteins, we demonstrated culture heterogeneity under industrially-relevant conditions. In a complementary approach, using RNAseq, we identified more than 200 small RNA molecules and improved the annotation of the model L. lactis, strain MG1363.
Another level of heterogeneity in cheese starter-cultures is their strain diversity. Complex, undefined starter cultures are very robust in production processes and their final product-properties are mostly superior to those derived from less-complex starter cultures. We study population dynamics and use genome-scale metabolic models and experimental evolution of mixed-cheese starter cultures to understand the underlying microbial interactions necessary for their functionality. Recent results showed that bacteriophages are vital to sustaining biodiversity in mixed dairy-starter cultures.
Another topic within this project is the physical interaction of microbial cells with matrix components, in cheese and fermented milk. The detailed characterisation and alteration of lactococcal surface properties, in an isogenic bacterial background, facilitated our study of the influence of surface properties on starter-culture functionalities.
This work will support the dairy industry in the development of healthier and tastier fermented products.
|Scientific papers in peer-reviewed journals||2015 Metabolism at evolutionary optimal States||View summary|
|Posters||2015 ArgR and its 3’UTR ArgX both regulate arc by influencing transcription and RNA stability/translation respectivel|
|Posters||2014 Metabolic modeling of a single Leuconostoc mesenteroides and seven Lactococcus lactis strains identifies the possible metabolic dependencies within a complex bacterial starter culture.|
|Lectures||2015 Functionality of multi-level diversity in complex fermentation starters|
This project developed generic models to predict the survival and subsequent growth of microorganisms that negatively impact food safety and quality. The models facilitate the development of strategies to ensure the safety and stability of products and the reputations of producers.
The growth characteristics of 20 different Listeria monocytogenes and 20 Lactobacillus plantarum strains were assessed under conditions of different temperature, pH, water activity and lactic-acid concentrations. This allowed for establishing the phenotypic variability using growth models. Experimental, biological and strain variation was quantified. The impact of strain and biological variability on the growth kinetics were comparable and both were higher than experimental variability. The cardinal parameters and their variability will be used as inputs for the gamma model to predict growth in food matrices. In addition, heat inactivation of the strains was assessed and D-values were determined and compared with data available in the literature. For thermal inactivation kinetics, the impact of strain variability was much higher than biological variability. Genomes of all twenty L. monocytogenes strains were sequenced and gene-trait-matching analyses are ongoing. Furthermore, the model was validated in milk and ham to quantify the impact of food-product composition. The comparison is expected to provide knowledge of the factors influencing variability.
Stable acid-resistant variants of Listeria monocytogenes were isolated and their resistance to a range of other types of stresses was tested. To identify a potential genetic basis for certain resistance phenotypes, a cluster was made based on these phenotypes, showing a large group (11 variants) that clustered together. A common gene trait in this cluster of variants was an SNP mutation (located in rpsU). A ΔrpsU deletion mutant is under construction to confirm the role of rpsU in stress resistance. To determine whether stable resistant variants can also be found in other organisms, other L. monocytogenes strains and mutants of B. subtilis and L. plantarum were investigated. All data will be used for simulation of population dynamics (sensitive and resistant population fraction) along a model food chain. An extra deliverable, in cooperation with the Biofilms project, has been set up in which the performance of L. monocytogenes acid-resistant variants in mixed biofilms with Lactobacillus plantarum has been evaluated.
For spore-forming bacteria, the variability in growth and heat inactivation of spores is also being assessed for 20 strains of Bacillus subtilis, Bacillus cereus and Geobacillus stearothermophilus. Experimental work on heat inactivation and growth of B. subtilis and B. cereus has been completed. For Geobacillus stearothermophilus strains, optimal media have been established to ensure recovery after heat treatments and spores have been harvested for 20 strains in 2 independent experiments. The data structure for fitting of the inactivation and growth model has been designed and prediction of variability of B. subtilis spore inactivation has been completed, showing significant strain variability. The experimental data was used to predict variability in out-growth of spore-formers and heat inactivation of their spores.
Other activities involving spore-forming bacteria include the generation of an overview of all genes involved in sporulation and germination of B. subtilis. This overview has been generated and captured in a database made accessible through SporeWeb. This was extended by adding gene-expression data acquired through the sporulation of wild type B. subtilis strains. In addition, the contribution of specific factors to spore-germination efficiency, in response to various heat treatments, was studied. The resulting data were used as input for a generic model describing the contribution of specific nutrient-receptor complexes in germination responses as a function of heat.
|Scientific papers in peer-reviewed journals||2015 Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein||View summary|
|Scientific papers in peer-reviewed journals||2015 Performance and resistance of Listeria monocytogenes wild type and multiple stress resistant variants in mixed culture biofilms with Lactobacillus plantarum||View summary|
|Scientific papers in peer-reviewed journals||2015 Diversity of acid stress resistan tvariants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU||View summary|
Many gram-positive bacteria can form spores that ensure survival under extreme conditions. Spores can survive adverse conditions and remain dormant for a long time, but germination and outgrowth can occur under favourable conditions. Current food-preservation techniques can fail to kill sufficient numbers of bacterial spores present in raw ingredients. When viable spores remain and eventually germinate, the vegetative cells can grow out in the final product, resulting in food spoilage, reduced shelf-life and possible food poisoning.
This project aims to broaden our knowledge of spore resistance and spore germination and outgrowth, using not only model Bacillus strains but also a wide variety of industrially-relevant spore formers. These strains are being studied at phenotypic and genomic levels; the genome sequences of 51 spore-forming Bacilli have been determined and are being analysed in relation to key spore properties.
Spore properties and germination behaviour of spores are determined during the sporulation process. Once resistant phase-bright spores are formed, they can remain dormant for prolonged periods of time. Upon sensing germinants in their environment, spores can germinate and vegetative cells can emerge, followed by outgrowth if conditions are favourable. Heterogeneous expression of genes that play important roles in germination were studied at the single cell level using time-lapse microscopy. This work led to interesting observations, such as premature spore lysis and heterogeneous promoter activity, which were studied in more detail. Importantly, this work also showed that the origin of germination heterogeneity could not be traced back to heterogeneity in gene expression, but that this is the result of heterogeneity in downstream processes, such as translation, modification and/or localisation of germination proteins.
In addition, spore superdormancy is being studied in this project. Superdormant spores are defined as dormant spores that fail to respond to germinants/nutrients that normally activate germinant receptors (these receptors play a key role in initiation of germination). Such spores can be present amongst spores that germinate well in heterogeneous spore populations. To quantify spore superdormancy, a pipeline was set up using a novel flow-cytometry approach, in which the responsiveness of superdormant spores to nutrient and non-nutrient germination triggers can be assessed. The phenotypes related to superdormancy were assessed for 20 B. cereus industrial isolates and significant phenotypical diversity was observed amongst these strains. Linking the genome sequences of these strains with phenotypic trait-relevant spore characteristics provides an excellent opportunity to extend our knowledge of these processes – from a limited number of reference strains to industrial isolates.
When dormant spores sustain damage, repair of such damage upon germination can be required, prior to outgrowth. The contribution of different recovery conditions (which include various food matrices) to the ability of sub-lethally injured spores to grow out, have been identified. In addition, transcriptome analyses, during germination, emergence of the vegetative cell and subsequent outgrowth, have been performed, yielding new target genes that could be involved in these processes. To extrapolate the observations related to the effects of sub-lethal treatments of model strains to industrial isolates, the characterisation of a range of spores of B. cereus industrial isolates is on-going.
Another important property of spores is their heat resistance, which is also being studied. Within the species B. subtilis, spore heat-resistance was assessed for a large number of strains. Two distinct groups could be identified, with spores from the highly heat-resistant group requiring more-intense heating for inactivation than the low-heat-resistant group. The genome sequences of these strains were analysed and, by performing gene-trait matching, a mobile genetic element was identified which was specifically present in the genomes of strains with high spore heat-resistance. It was demonstrated that this element was responsible for the increased heat-resistance of spores. This element is not restricted to B. subtilis and is found in many other spore- forming Bacilli, indicating a role in evolution and adaptation of these strains. This study provides insight into prediction of spore heat-inactivation processes and the mechanism by which increased spore heat-resistance is mediated and will, ultimately, allow for better detection and control of highly heat-resistant bacterial spores in foods.
The last component of the Spores project translates knowledge gained in model spore-forming organisms to wild-type food strains. The genomic content of 47 Bacillus spp. food isolates (which cause problems to industry) was determined via whole-genome sequencing, contig assembly and gene annotation. For selected strains (13 B. subtilis and 5 B. thermoamylovorans) a phenotypic characterisation of germination properties, under selected conditions, was performed and significant differences between the strains of each species were observed. Subsequent work will include studying identified genetic factors to validate their suggested roles in the phenotype with which they are associated.
|Scientific papers in peer-reviewed journals||2015 Draft genome sequences of four Bacillus thermoamylovorans strains, isolated from milk and acacia gum, a food ingredient||View summary|
|Scientific papers in peer-reviewed journals||2015 Next generation whole genome sequencing of eight strains of Bacillus cereus, isolated from food||View summary|
|Scientific papers in peer-reviewed journals||2016 Bacterial spores in food: survival, emergence and outgrowth||View summary|
|Scientific papers in peer-reviewed journals||2015 T-Rex: Transcriptome analysis webserver for RNA-seq Expression data||View summary|
Most microorganisms have the capacity to grow in a surface-attached state, in which they live as multicellular communities embedded in an extracellular polymeric matrix. These biofilms provide the environment for microorganisms to display altered behaviour compared to their planktonic lifestyle. Biofilms might increase the cell-resistance to antimicrobials as they can function as protective barriers against the environment. Moreover, microenvironments provided within the biofilm might increase heterogeneity within the population. This variety in behaviour complicates control of microbial contaminants. This project will provide mechanistic insights into the factors contributing to biofilm formation and persistence in processing environments, aiming to enhance control of both biofilm formation and (re)contamination of food products.
The project is organised into four sub-projects targeting biofilms encountered in different food-related industrial environments: Lactobacillus biofilms, biofilms of thermophilic and mesophilic spore formers, and mixed-species biofilms.
In the subproject Lactobacillus biofilms (WP1), the biofilm-forming capacity has been assessed for Lactobacillus plantarum food isolates obtained from industrial partners and for the model strain L. plantarum WCFS1. Mechanisms involved in biofilm formation have been studied by using available L. plantarum mutant strains in genes affecting cell-surface characteristics. Genome sequences of 6 L. plantarum isolates have been exploited for analysis of genomic differences and similarities between L. plantarum strains relating to biofilm formation. The project further aims to quantify the competitive population dynamics of L. plantarum isolates in multi-strain communities in liquid culture and on surfaces in static and continuous-flow biofilms. With this activity we aim to construct an interaction network describing biofilm ecology and relevant features for performance and persistence in a genetic make-up of multi-strain communities.
Biofilm formation of Bacillus cereus reference strains ATCC 14579 and ATCC 10987 and 21 undomesticated food isolates was studied on polystyrene and stainless steel in WP2 (Bacillus cereus biofilms). Results show that stainless steel, as a contact material, provides more-favourable conditions for B. cereus biofilm formation and maturation compared to polystyrene. This effect could possibly be linked to iron availability, as we show that free-iron availability affects B. cereus biofilm formation.
Evidence was provided for a pleiotropic role of rpoN in B. cereus, supporting its adaptive response and survival in a range of conditions including biofilm formation.
The sporulation dynamics in dry and wet biofilms and heat resistance of produced spores was analysed and compared to planktonic growth conditions.
WP3 has focussed on characterisation of thermophilic spore formers. Three isolates – (Geobacillus thermoglucosidans, Geobacillus stearothermophilus and Anoxybacillus flavithermus) – previously isolated as the most abundant and most potent biofilm-forming strains isolated from dairy factory industrial samples – have been characterised in detail with respect to their growth conditions for biofilm formation and spore resistance. Genome sequences of 7 thermophilic industrial isolates have been determined and are subject to genome mining for traits related to growth in a dairy environment and for construction of a biofilm gene network. Gene expression profiles, under selected biofilm conditions and in different biofilm forming stages, have been determined
In WP4, the formation of single and mixed species biofilms of L. monocytogenes EGD-e and Lactobacillus plantarum WCFS1 as secondary species was characterised at different temperatures and medium compositions (nutrient-rich medium with and without supplementary manganese, glucose and salt). Also the role of sigB, which encodes a major transcriptional regulator of stress-response genes, in single and mixed species biofilm formation was explored.
WP5 focusses on assessing the physiological state of model spoilage microorganisms and their (out)growth potential, assessed at single cell or spore level in the absence and presence of selected (combined) preservation stresses. Flow cytometry and single-cell sorting was used to study the heterogeneity in outgrowth of B. cereus spores isolated from biofilms. For a second deliverable, sigB (general stress) and KatA (oxidative stress) fluorescent reporters have been investigated as potential (bio)markers to study different physiological states of B. subtilis and B. cereus under stress and linked to outgrowth potential.
|Scientific papers in peer-reviewed journals||2015 Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores||View summary|
|Scientific papers in peer-reviewed journals||2015 Impact of growth conditions and role of sigB on L. monocytogenes fitness in single and mixed biofilms cultured with L. plantarum WCFS1||View summary|
|Scientific papers in peer-reviewed journals||2016 Sporulation dynamics and spore heat resistance in wet and dry biofilms of Bacillus cereus||View summary|
|Scientific papers in peer-reviewed journals||2015 Bacillus cereus ATCC 14579 RpoN (Sigma 54) is a pleiotropic regulator of growth, carbohydrate metabolism, motility, biofilm formation and toxin production||View summary|
Project leader: Dr Bart Keijser
Time frame 2012 – 2016
Project code: OH001
Project login (project members only)
This project aims to identify the biological processes in the oral ecosystem responsible for maintaining oral health. The project is based on the hypothesis that ‘oral health’ is the ability of the oral ecosystem to adapt to and counteract stresses, where the oral ecosystem is defined as the tripartite of oral microbiota, saliva and host (mucosal) immunity. The project comprises five interconnected work packages: a sixth work package is focussed on knowledge dissemination.WP1 aims to provide a detailed description of the oral ecosystem, including microbial, biochemical, immunological and metabolic parameters. In WP2, short-term intervention challenges will be applied to gain understanding of the dynamic interplay in the oral ecosystem following a perturbance. In WP3 a systems-biology network model will be built using data derived from Work Packages 1 and 2. In WP4, in vitro biofilm models will be used to explore the interactions between bacteria within an oral biofilm and interactions between salivary components and the microbial biofilm. WP5 aims to identify the interplay between saliva, oral microbial components and the host cells that line the oral cavity: the gingiva, tongue and/or buccal side. This will be achieved via high-content high-throughput microscopy. The in vitro studies will provide mechanistic insight into microbiota-host-saliva interactions and could support the biomarkers identified in WPs 1-3. The importance of knowledge dissemination is emphasised by the inclusion of a separate work package (WP6) that specifically focuses on translating scientific insights for (future) healthcare professionals, the general public, legislators and policy makers.
During the previous two years, a clinical evaluation study was completed (WP1) which assessed and identified the boundaries of a healthy oral ecosystem. This study involved 268 healthy volunteers. Via the study we established an extensive set of clinical (oral) health parameters, including gingival bleeding and caries levels. Furthermore, samples were collected for an extensive assessment of the composition of the oral ecosystem, including:
- microbiota analysis of saliva, interproximal plaque, supragingival and subgingival plaque and the posterior and anterior tongue biofilms
- concentration range of 10 salivary proteins with known relevance to oral health
- salivary peptide profiles, via MALDI-TOF analysis
- the salivary metabolome. levels and function of oral polymorphonuclear neutrophilic granulocytes (oPMNs).
Through application of advanced, statistical, machine-learning techniques we identified subgroups of healthy individuals that differed in their microbial, biochemical or metabolic oral profiles. Work is ongoing to provide better understanding of the functional significance of these differences.
In parallel, in vitro models have been established for the dental microbial biofilm as well as mucosal epithelial interactions. We were able to show that exposure of the dental biofilm to a particular component – that interferes with microbial communication (quorum sensing) – eliminated lactate production of oral biofilms. The findings have been patented. Also, we have been able to establish a high-throughput in vitro wound-healing assay using automated microscopy, that allows for qualitative description of the kinetics of oral-wound repair. Using this technique we were able to confirm earlier findings that an oral pathogen inhibited wound closure. We also showed that a related, commensal species enhanced wound closure.
We are currently performing a clinical intervention study to explore the dynamic (biochemical, microbial and immunological) interactions of the oral ecosystem upon perturbance and their involvement in maintenance of oral health.
|Scientific papers in peer-reviewed journals||2015 Personalized microbial network inference via co-regularized spectral clustering||View summary|
|Scientific papers in peer-reviewed journals||2015 In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms||View summary|
||2014 The oral microbiome|
|Invited lectures||2014 Saliva and the oral mircobiome|