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Pulsed electric field pre-treatment can be used for loading sugars into living cells, which may make cells more robust during subsequent spray drying. In this study we investigated the effect of the (change of) drying medium after PEF pre-treatment with trehalose for its impact on survival during subsequent spray drying for L. plantarum WCFS1. Our results showed that when trehalose was used as drying medium, no difference in survival with and without PEF pre-treatment was observed. However, when reconstituted skim milk and maltodextrin DE19 were used as drying medium an increased survival of the PEF-treated culture was observed compared to the control.

The production of healthy, nutritious, tasty, and safe foods requires efficient strategies to control foodborne pathogens along the food chain. One of these pathogens is the notorious foodborne Listeria monocytogenes. L. monocytogenes is a robust, ubiquitously present human pathogen, and the cause of life-threatening listeriosis in the very young, elderly, pregnant, and immunocompromised persons, the so-called YOPI population. The incidence of L. monocytogenes infections is low, but the severity of listeriosis and the high mortality rate rank it among the top three causes of death by foodborne disease.There is an inherent heterogeneity in microbial populations, and this heterogeneity gives L. monocytogenes the capacity to cope with stresses during transmission from the environment to the human host. Stochastic differences in stress response between individual cells of a population, lead to the differential survival of cells after lethal stresses such as heat or low pH, and ultimately result in tailing of the inactivation curve. The heterogeneity in a population can be either transient, where certain cells temporarily have different properties, or stable, where individual cells have undergone genetic changes that make them better able to resist (lethal) stress. Cells with genetic changes are called stable variants, and can be isolated from the tail of the inactivation curve.    Almost all research that has been done with L. monocytogenes has focussed on the diversity that is already present in a population. Therefore, in chapter 2, we investigated the rate at which new diversity is generated by mutations. Using a high-throughput protocol, we have experimentally determined the mutation rate of 20 L. monocytogenes strains, and found a mutator strain with an insertion in the DNA mismatch repair gene mutS, that resulted in a 100-1000-fold increase in mutation rate. To our knowledge, this is the first mutator strain of L. monocytogenes isolated from fo

It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus L-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as

Bacteria are commonly used in food industry as starter cultures or probiotic supplements. Production of these bacterial food ingredients requires growth of the bacteria followed by storage until they are used as fermentation starter or ingested as probiotic. To prolong the shelf life of these bacteria, drying processes such as freeze drying and spray drying are used to dry these ingredients into a powder. A drawback of drying is that the viability of the dried bacterial cultures decrease along the process chain. Therefore, many studies focus on improving the survival of these cultures during drying and subsequent storage. Often the bacteria are dried in a carrier matrix consisting of carbohydrates and/or proteins to increase their survival. Some studies also suggest that increased intracellular concentrations of protective solutes such as for example trehalose can also result in better survival after processing. Trehalose is a disaccharide that is accumulated intracellularly by several organisms, e.g. bacteria, yeasts and plants, under desiccation stress. Intracellular trehalose concentrations can also be increased by a mechanical method, i.e. pulsed electric field treatment. Pulsed electric field (PEF) treatment is the application of short high voltage pulses to a product between two electrodes which can result in permeabilization of the cellular membrane. PEF pre-treatment leading to increased intracellular trehalose concentrations has been shown to enhance robustness of mammalian cells towards processing, such as cryopreservation. The aim of this thesis was to develop a PEF pre-treatment leading to increased intracellular protective solutes concentrations in bacteria in order to enhance survival during drying and subsequent powder storage. This aim was divided in two parts. First, to develop a PEF pre-treatment leading to increased intracellular trehalose concentrations, while maintaining culture viability. And secondly, to evaluate whether this PEF pre-treatment

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods. The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species. Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively. Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant. A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after o

Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.

A major drawback to the industrial application of many biocolorants is their instability to processing conditions, thereby limiting their use to replace artificial colorants. 3-Deoxyanthocyanidins have promising features to ensure colour stability in food processing conditions. This study evaluated the stability of apigeninidin, the main 3-deoxyanthocyanidin from sorghum leaf sheaths, to food processing conditions in watery extracts and in a maize porridge. Apigeninidin was not soluble at pH 5.04 ± 0.02. However, apigeninidin was soluble and stable at pH 6–10 with increased colour density and resistance to bleaching at alkaline pH. A heat treatment of 121 °C/30 min degraded 61% of the anthocyanins. At 65 °C, degradation rate of apigeninidin was four times lower at pH 9.03 ± 0.04 than 6.08 ± 0.02. Storage at room temperature promoted endothermic degradation reactions. Nevertheless, photodegradation of apigeninidin was not observed during storage. In the maize porridge, thermal stability of apigeninidin and redness were similar at pH 4–6 whereas they were higher at pH 9.03 ± 0.04. In summary, the watery extract of apigeninidin from sorghum leaf sheaths showed good stability regarding common industrial processes. Nevertheless, the biocolorant's precipitation at pH 5.04 ± 0.02 and degradation at pH 6.08 ± 0.02 and 9.03 ± 0.04 need further investigation to optimise its industrial applications.

Pulsed electric field (PEF) processing has been developed and applied in food industry for several purposes. In this study we used PEF for increasing the intracellular trehalose content in Lactobacillus plantarum WCFS1. Our results indicated that it is possible to increase intracellular trehalose content in Lactobacillus plantarum WCFS1 to ~100 mM with 75% survival when applying a PEF treatment with an electric field strength of 7.5 kV/cm. Fluorescence staining of PEF-treated cells with propidium iodide (PI) and SYTO 9 showed that at 7.5 kV/cm only a small fraction (23%) of the cells had a permeated membrane by this PEF treatment, of which approximately half had an irreversible permeated membrane. Resealing of the pores in the membrane for PI uptake was very fast, in the order of seconds. These results indicate that PEF treatment is promising for increasing intracellular trehalose, but further optimization is required to increase the trehalose content in all cells.

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed differential expression of 319 genes of which 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants.

Prenylated phenolic compounds, i.e. those bearing a C5-isoprenoid (prenyl) substituent, are abundant in plants from the Fabaceae (legume) family and are potential natural antibacterial agents against resistant pathogenic bacteria. To understand the antibacterial properties of these compounds, (quantitative) structure-activity relationships and mode of action of these molecules were investigated against Gram positive and negative bacteria. Compounds belonging to the flavonoid, isoflavonoid and stilbenoid classes were studied. Antibacterial activity was modulated by the (sub)class of phenolic compound, as well as by the configuration, position and number of prenyl groups. Prenylated isoflavones were found to be better antibacterials than prenylated pterocarpans and prenylated stilbenoids. It was also shown that chain prenylation increased the antibacterial activity more than pyran-ring prenylation. Diprenylated compounds were among the most active antibacterials with minimum inhibitory concentrations of less than 10 µg/mL against Listeria monocytogenes. The main molecular characteristics defining antibacterial activity were molecular shape (including flexibility and globularity) and hydrophobicity. Regarding the mode of action of these compounds, it was shown that prenylated phenolic compounds can disrupt the integrity of the membrane by permeabilization very quickly. Interestingly, some good antibacterial prenylated (iso)flavonoids showed good permeabilization capacity whereas others not (including diprenylated molecules), highlighting potential differences in their interactions with the bacterial membrane. Likewise, it was shown that Gram negative intrinsic resistance towards prenylated phenolic compounds is primarily due to the activity of efflux pump systems and that it can be overcome by using an efflux pump inhibitor in combination with antibacterial prenylated compounds. Last, in vitro production of prenylated phenolic compounds was performed with microbial pre

Mutandabota is a dairy product consumed as a major source of proteins and micronutrients in Southern Africa. In this study the microbial safety of traditional and a variant of mutandabota fermented with the probiotic Lactobacillus rhamnosus yoba (yoba mutandabota) was investigated by challenging the products with five important food pathogens: Listeria monocytogenes, Salmonella spp., Campylobacter jejuni, Escherichia coli O157:H7 and Bacillus cereus. Pasteurized full-fat cow's milk was used for producing traditional and yoba mutandabota, and was inoculated with a cocktail of strains of the pathogens at an inoculum level of 5.5 log cfu/mL. Survival of the pathogens was monitored over a potential consumption time of 24 h for traditional mutandabota, and over 24 h of fermentation followed by 24 h of potential consumption time for yoba mutandabota. In traditional mutandabota (pH 3.4 ± 0.1) no viable cells of B. cereus and C. jejuni were detected 3 h after inoculation, while L. monocytogenes, E. coli O157:H7 and Salmonella spp. significantly declined (P <0.05), but could still be detected (<3.5 log inactivation) at the end of the potential consumption time. This indicated that consumption of traditional mutandabota exposes consumers to the risk of food-borne microbial infections. In yoba mutandabota, L. rhamnosus yoba grew from 5.5 ± 0.1 log cfu/mL to 9.1 ± 0.4 log cfu/mL in the presence of pathogens. The pH of yoba mutandabota dropped from 4.2 ± 0.1 to 3.3 ± 0.1 after 24 h of fermentation, mainly due to organic acids produced during fermentation. Only Salmonella spp. was able to grow in yoba mutandabota during the first 9 h of fermentation, but then decreased in viable plate count. None of the tested pathogens were detected (> 3.5 log inactivation) after 3 h into potential consumption time of yoba mutandabota. Inactivation of pathogens in mutandabota is of public health significance because food-borne pathogens endanger public health upon consumption of contaminated foo

This study aimed at determining bacterial concentration and diversity in fresh tropical shrimps (. Penaeus notialis) and their surrounding brackish waters and sediment. Freshly caught shrimp, water and sediment samples were collected in Lakes Nokoue and Aheme in Benin (West Africa) during two periods with different water salinity and temperature. We used complementary culture-dependent and culture-independent methods for microbiota analysis. During both sampling periods, total mesophilic aerobic counts in shrimp samples ranged between 4.4 and 5.9 log CFU/g and were significantly higher than in water or sediment samples. In contrast, bacterial diversity was higher in sediment or water than in shrimps. The dominant phyla were Firmicutes and Proteobacteria in shrimps, Firmicutes, Proteobacteria, and Actinobacteria in water, and Proteobacteria and Chloroflexi in sediment. At species level, distinct bacterial communities were associated with sediment, water and shrimps sampled at the same site the same day. The study suggests that the bacterial community of tropical brackish water shrimps cannot be predicted from the microbiota of their aquatic environment. Thus, monitoring of microbiological quality of aquatic environments might not reflect shrimp microbiological quality.

This review elucidates the state-of-the-art knowledge about pathogen population heterogeneity and describes the genotypic and phenotypic analyses of persister subpopulations and stress-resistant variants. The molecular mechanisms underlying the generation of persister phenotypes and genetic variants are identified. Zooming in on Listeria monocytogenes, a comparative whole-genome sequence analysis of wild types and variants that enabled the identification of mutations in variants obtained after a single exposure to lethal food-relevant stresses is described. Genotypic and phenotypic features are compared to those for persistent strains isolated from food processing environments. Inactivation kinetics, models used for fitting, and the concept of kinetic modeling-based schemes for detection of variants are presented. Furthermore, robustness and fitness parameters of L. monocytogenes wild type and variants are used to model their performance in food chains. Finally, the impact of stress-resistant variants and persistence in food processing environments on food safety is discussed.

Food chains are susceptible to contaminations from food-borne hazards, including pathogens and chemical contaminants. An assessment of the potential product-hazard combinations can be supported by using multiple data sources. The objective of this study was to identify the main trends of food safety hazards in the European spice and herb chain, and then, evaluate how the data sources can be used during each step of a microbiological and a toxicological risk assessment. Thereafter, the possibilities and limitations of the selected data sources for the risk assessment of certain hazards in spices and herbs are examined. European governmental alerting and monitoring data and legislation were examined and evaluated for particular product-hazard combinations. Pathogenic microorganisms, particularly Salmonella spp. and pathogenic Bacillus spp., were identified as a potential concern in black pepper and dried herbs, while mycotoxins like aflatoxin (B1) and ochratoxin A were a probable concern in chilies (including chili powder and cayenne), paprika, and nutmeg. Evaluating multiple, accessible, data sources can support several steps during the risk assessment process as seen for the hazard identification step. Therefore, identifying the potential spice and herb food safety hazards in the chain and other specific data can support risk assessors in compiling a comprehensive risk assessment.

The spoilage activity of Pseudomonas psychrophila and Carnobacterium maltaromaticum, two tropical shrimp (Penaeus notialis) spoilage organisms, was assessed in cooked shrimps stored at 0 to 28 °C. Microbiological, chemical and sensory analyses were performed during storage. P. psychrophila had a higher growth rate and showed a higher spoilage activity at temperatures from 0 to 15 °C, while at 28 °C, C. maltaromaticum had a higher growth rate. The spoilage activity of P. psychrophila was found to be higher in cooked shrimp than in fresh shrimp. Observed shelf-life data of shrimps stored at constant temperatures were used to validate a previously developed model that predicts tropical shrimp shelf-life at constant storage temperatures. Models predicting the growth of the spoilage organisms as a function of temperature were constructed. The validation of these models under dynamic storage temperatures simulating temperature fluctuation in the shrimp supply chain showed that they can be used to predict the shelf-life of cooked and fresh tropical shrimps.

Maintaining the freshness of shrimp is a concern to shrimp stakeholders. To improve shrimp quality management, it is of importance to evaluate shrimp spoilage characteristics. Therefore, microbiological, sensory, and chemical changes of naturally contaminated tropical brackish water shrimp (Penaeus notialis) during storage at 28 °C, 7 °C and 0 °C were assessed. H2S-producing bacteria were the dominant group of microorganisms at 28 °C and 7 °C whereas Pseudomonas spp. were dominant at 0 °C. Total volatile basic nitrogen and trimethylamine correlated well (R2 > 0.90) with the sensory scores. An empirical model to predict the shelf-life of naturally contaminated tropical shrimp as a function of storage temperature was developed. Specific groups of organisms were isolated at the sensory rejection times and assessed for spoilage potential in shrimps of which the endogenous flora was heat inactivated. Isolates capable of producing strong off-odor identified by 16S rRNA sequencing were mainly lactic acid bacteria (LAB) and Enterobacteriaceae at 28 °C or 7 °C and Pseudomonas spp. and LAB at 0 °C. The study contributes to the knowledge about tropical shrimp spoilage and provides a basis for the development of methods and tools to improve shrimp quality management. Keywords: Shrimp quality; Microbiological change; TVBN; Sensory rejection; Shelf-life prediction

The dynamic response of microorganisms to environmental conditions depends on the behavior of individual cells within the population. Adverse environments can select for stable stress resistant subpopulations. In this study, we aimed to get more insight in the diversity within Listeria monocytogenes LO28 populations, and the genetic basis for the increased resistance of stable resistant fractions isolated after acid exposure. Phenotypic cluster analysis of 23 variants resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation, and virulence indicators. A higher glutamate decarboxylase activity correlated with increased acid resistance. Whole genome sequencing revealed mutations in rpsU, encoding ribosomal protein S21 in the largest phenotypic cluster, while mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and high hydrostatic pressure resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants. The finding that acid stress selects for rpsU variants provides potential insights in the mechanisms underlying population diversity of L. monocytogenes.

In food safety management, sampling is an important tool for verifying control. Sampling by nature is a stochastic process. However, uncertainty regarding results is made even greater by the uneven distribution of microorganisms in a batch of food. This article reviews statistical aspects of sampling and describes the impact of distributions on the sampling results. Five different batch contamination scenarios are illustrated: a homogeneous batch, a heterogeneous batch with high- or low-level contamination, and a batch with localized high- or low-level contamination. These batch contamination scenarios showed that sampling results have to be interpreted carefully, especially when heterogeneous and localized contamination in food products is expected.

In recent years, the Beninese shrimp sector has faced a ban on export to the European Union due to lack of compliance with food safety standards. The present study aimed at obtaining insight into the factors that determine shrimp quality and safety in Benin. A survey was conducted to investigate the relationships between stakeholders, the conditions under which shrimps are handled at fishing areas and processed at shrimp plants, and the stakeholders' perceptions of quality. A total of 325 fishermen, 128 intermediate traders, 12 collectors, and 3 shrimp processing plant managers were interviewed face to face. The results showed that various specific relations existed between the stakeholders. For example, loyalty was ensured by family relationships, or incentives were provided to ensure a supply of shrimps between stakeholders. Shrimp handling practices during the steps prior to shrimp processing at the plants were not in agreement with the requirements of the European regulations. For example, shrimps were kept at ambient temperature (28 ± 1°C) by 94.1% of fishermen and 60.9% of intermediate traders. Shrimps were also stored in inappropriate holding containers and washed with nonpotable water. Fishermen, intermediate traders, and collectors considered shrimp size and texture their priority quality attributes, whereas plant managers considered shrimp appearance (freshness) and texture their priority quality attributes. This survey demonstrated that the steps prior to shrimp processing at the plants are the critical steps for shrimp quality and safety because of temperature abuse and inappropriate hygienic conditions. There is a need to communicate and provide incentives for the stakeholders in the first part of the chain to give priority to shrimp freshness. Moreover, training in Good Fishing Practices and safe food handling practices and evaluation of compliance with the practices through monitoring will contribute to better shrimp quality and safety management.

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.

The ability of microorganisms to adapt to changing environments and gain cell robustness, challenges the prediction of their history-dependent behaviour. Using our model organism Bacillus cereus, a notorious Gram-positive food spoilage and pathogenic spore-forming bacterium, a strategy will be described that allows for identification of biomarkers for robustness. First an overview will be presented of its two-component systems that generally include a transmembrane sensor histidine kinase and its cognate response regulator, allowing rapid and robust responses to fluctuations in the environment. The role of the multisensor hybrid kinase RsbK and the PP2C-type phosphatase RsbY system in activation of the general stress sigma factor sB is highlighted. An extensive comparative analysis of transcriptional landscapes derived from B. cereus exposed to mild stress conditions such as heat, acid, salt and oxidative stress, revealed that, amongst others sB regulated genes were induced in most conditions tested. The information derived from the transcriptome data was subsequently implemented in a framework for identifying and selecting cellular biomarkers at their mRNA, protein and/or activity level, for mild stressinduced microbial robustness towards lethal stresses. Exposure of unstressed and mild stress-adapted cells to subsequent lethal stress conditions (heat, acid and oxidative stress) allowed for quantification of the robustness advantage provided by mild stress pretreatment using the plate-count method. The induction levels of the selected candidate-biomarkers, sB protein, catalase activity and transcripts of certain proteases upon mild stress treatment, were significantly correlated to mild stress-induced enhanced robustness towards lethal thermal, oxidative and acid stresses, and were therefore suitable to predict these adaptive traits. Cellular biomarkers that are quantitatively correlated to adaptive behavior will facilitate our ability to predict the impact of adap

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 o

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.

Short- and long-term exposure to mild stress conditions can activate stress adaptation mechanisms in pathogens, resulting in a protective effect toward otherwise lethal stresses. The mesophilic strains Bacillus cereus ATCC 14579 and ATCC 10987 and the psychrotolerant strain B. weihenstephanensis KBAB4 were cultured at 12°C and 30°C until the exponential growth phase (i) in the absence of salt, (ii) in the presence of salt, and (iii) with salt shock after they reached the exponential growth phase and subsequently heat inactivated. Both the first-order model and the Weibull model were fitted to the inactivation kinetics, and statistical indices were calculated to select for each condition the most appropriate model to describe the inactivation data. The third-decimal reduction times (which reflected the times needed to reduce the initial number of microorganisms by three decimal powers) were determined for quantitative comparison. The heat resistance of both mesophilic strains increased when cells were salt cultured and salt shocked at 30°C, whereas these salt-induced effects were not significant for the psychrotolerant strain. In contrast, only the psychrotolerant strain showed salt-induced heat resistance when cells were cultured at 12°C. Therefore, culturing temperature and strain diversity are important aspects to address when adaptive stress responses are quantified. The activated adaptive stress response had an even larger impact on the number of surviving microorganisms when the stress factor (i.e., salt) was still present during inactivation. These factors should be considered when stress-integrated predictive models are developed that can be used in the food industry to balance and optimize processing conditions of minimally processed foods

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Ou

Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions. B. cereus continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na+/H+, and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms

Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log10-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.

The food-borne pathogen Bacillus cereus can acquire enhanced thermal resistance through multiple mechanisms. Two Bacillus cereus strains, ATCC 10987 and ATCC 14579, were used to quantify the effects of salt stress and physiological state on thermotolerance. Cultures were exposed to increasing concentrations of sodium chloride for 30 min, after which their thermotolerance was assessed at 50°C. Linear and nonlinear microbial survival models, which cover a wide range of known inactivation curvatures for vegetative cells, were fitted to the inactivation data and evaluated. Based on statistical indices and model characteristics, biphasic models with a shoulder were selected and used for quantification. Each model parameter reflected a survival characteristic, and both models were flexible, allowing a reduction of parameters when certain phenomena were not present. Both strains showed enhanced thermotolerance after preexposure to (non)lethal salt stress conditions in the exponential phase. The maximum adaptive stress response due to salt preexposure demonstrated for exponential-phase cells was comparable to the effect of physiological state on thermotolerance in both strains. However, the adaptive salt stress response was less pronounced for transition- and stationary-phase cells. The distinct tailing of strain ATCC 10987 was attributed to the presence of a subpopulation of spores. The existence of a stable heat-resistant subpopulation of vegetative cells could not be demonstrated for either of the strains. Quantification of the adaptive stress response might be instrumental in understanding adaptation mechanisms and will allow the food industry to develop more accurate and reliable stress-integrated predictive modeling to optimize minimal processing conditions.