Characterization and Exposure Assessment of Emetic Bacillus cereus and Cereulide Production in Food Products on the Dutch Market.

The emetic toxin cereulide, which can be produced by Bacillus cereus, can be the cause of food poisoning upon ingestion by the consumer. The toxin causes vomiting and is mainly produced in farinaceous food products. This article includes the prevalence of B. cereus and of cereulide in food products in The Netherlands, a characterization of B. cereus isolates obtained, cereulide production conditions, and a comparison of consumer exposure estimates with those of a previous exposure assessment. Food samples (n = 1,489) were tested for the presence of B. cereus; 5.4% of the samples contained detectable levels (>10(2) CFU/g), and 0.7% contained levels above 10(5) CFU/g. Samples (n = 3,008) also were tested for the presence of cereulide. Two samples (0.067%) contained detectable levels of cereulide at 3.2 and 5.4 µg/kg of food product. Of the 481 tested isolates, 81 produced cereulide and/or contained the ces gene. None of the starch-positive and hbl-containing isolates possessed the ces gene, whereas all strains contained the nhe genes. Culture of emetic B. cereus under nonoptimal conditions revealed a delay in onset of cereulide production compared with culture under optimal conditions, and cereulide was produced in all cases when B. cereus cells had been in the stationary phase for some time. The prevalence of cereulide-contaminated food approached the prevalence of contaminated products estimated in an exposure assessment. The main food safety focus associated with this pathogen should be to prevent germination and growth of any B. cereus present in food products and thus prevent cereulide production in foods.

Risk-based estimate of effect of foodborne diseases on public health, Greece.

The public health effects of illness caused by foodborne pathogens in Greece during 1996-2006 was quantified by using publicly available surveillance data, hospital statistics, and literature. Results were expressed as the incidence of different disease outcomes and as disability-adjusted life years (DALY), a health indicator combining illness and death estimates into a single metric. It has been estimated that each year ≈370,000 illnesses/million inhabitants are likely caused because of eating contaminated food; 900 of these illnesses are severe and 3 fatal, corresponding to 896 DALY/million inhabitants. Ill-defined intestinal infections accounted for the greatest part of reported cases and 27% of the DALY. Brucellosis, echinococcosis, salmonellosis, and toxoplasmosis were found to be the most common known causes of foodborne illnesses, being responsible for 70% of the DALY. Overall, the DALY metric provided a quantitative perspective on the impact of foodborne illness that may be useful for prioritizing food safety management targets.

Comparing nonsynergy gamma models and interaction models to predict growth of emetic Bacillus cereus for combinations of pH and water activity values.

This research aims to test the absence (gamma hypothesis) or occurrence of synergy between two growth-limiting factors, i.e., pH and water activity (a(w)), using a systematic approach for model selection. In this approach, preset criteria were used to evaluate the performance of models. Such a systematic approach is required to be confident in the correctness of the individual components of the combined (synergy) models. With Bacillus cereus F4810/72 as the test organism, estimated growth boundaries for the a(w)-lowering solutes NaCl, KCl, and glucose were 1.13 M, 1.13 M, and 1.68 M, respectively. The accompanying a(w) values were 0.954, 0.956, and 0.961, respectively, indicating that equal a(w) values result in similar effects on growth. Out of the 12 models evaluated using the preset criteria, the model of J. H. T. Luong (Biotechnol. Bioeng. 27:280-285, 1985) was the best model to describe the effect of a(w) on growth. This a(w) model and the previously selected pH model were combined into a gamma model and into two synergy models. None of the three models was able to describe the combined pH and a(w) conditions sufficiently well to satisfy the preset criteria. The best matches between predicted and experimental data were obtained with the gamma model, followed by the synergy model of Y. Le Marc et al. (Int. J. Food Microbiol. 73:219-237, 2002). No combination of models that was able to predict the impact of both individual and combined hurdles correctly could be found. Consequently, in this case we could not prove the existence of synergy nor falsify the gamma hypothesis.

Physiological parameters of Bacillus cereus marking the end of acid-induced lag phases.

During lag phases microbial cells adapt to their environment and prepare to proliferate. Physiological parameters of B. cereus cells upon exposure to near-growth-boundary acid stress were investigated and markers for the transition between lag phase and growth were identified using fluorescent probes combined with flow cytometry. Determination of cell counts and optical density revealed lag phases of 1h, 2h and 5h, in cultures shifted to pH 7, pH 5.3 (set with lactic acid) and pH 4.9 (set with sulfuric acid), respectively. The obtained lag phases fitted the trends in ATP levels, which were constant during the lag phase and increased after the onset of growth. Both the percentage of PI-stained cells and cells with a significant membrane potential decreased during the lag phase. This points to repair of membrane damage and the loss of membrane potential. However, both trends extended in the growth phase, thus not suitable to mark the onset of growth. The activity of the electron transfer chain and esterases did allow for assessment of transition between lag and growth phase. These activities were generally low during the lag phase and increased after the onset of growth. Our results show that, independent of the duration of the lag phase, for different conditions the same physiological trends could be observed. The change in signal of selected probes can be used as a marker for transition from lag phase to the growth phase and may aid in identification of novel targets interfering with bacterial exit from lag phase.

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard.

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.

Comparing nonsynergistic gamma models with interaction models to predict growth of emetic Bacillus cereus when using combinations of pH and individual undissociated acids as growth-limiting factors.

A combination of multiple hurdles to limit microbial growth is frequently applied in foods to achieve an overall level of protection. Quantification of hurdle technology aims at identifying synergistic or multiplicative effects and is still being developed. The gamma hypothesis states that inhibitory environmental factors aiming at limiting microbial growth rates combine in a multiplicative manner rather than synergistically. Its validity was tested here with respect to the use of pH and various concentrations of undissociated acids, i.e., acetic, lactic, propionic, and formic acids, to control growth of Bacillus cereus in brain heart infusion broth. The key growth parameter considered was the maximum specific growth rate, mu(max), as observed by determination of optical density. A variety of models from the literature describing the effects of various pH values and undissociated acid concentrations on mu(max) were fitted to experimental data sets and compared based on a predefined set of selection criteria, and the best models were selected. The cardinal model developed by Rosso (for pH dependency) and the model developed by Luong (for undissociated acid) were found to provide the best fit and were combined in a gamma model with good predictive performance. The introduction of synergy factors into the models was not able to improve the quality of the prediction. On the contrary, inclusion of synergy factors led to an overestimation of the growth boundary, with the inherent possibility of leading to underestimation of the risk under the conditions tested in this research.

Comparison of two optical-density-based methods and a plate count method for estimation of growth parameters of Bacillus cereus.

Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely, the 2-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. Values for the maximum specific growth rate (mu(max)) derived by the 2FD method and by the RRD method were of the same order of magnitude, but some marked differences between the two approaches were apparent. Whereas the 2FD method allowed the derivation of values for lag time (lambda) from the data, this was not possible with the RRD method. However, the RRD method gave many more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation but that the choice of method depends on the objectives of the research.

Occurrence of Enterobacter sakazakii in food production environments and households.

Enterobacter sakazakii occasionally causes illness in premature babies and neonates. Contamination of infant formulae during factory production or bottle preparation is implicated. Advice to health-care professionals focuses on bottle preparation, but the effectiveness of prevention depends on the degree of contamination and contamination sites, which are generally unknown. To keep contamination to a minimum in the finished product depends on knowledge of the occurrence of E sakazakii. We used a refined isolation and detection method to investigate the presence of this micro-organism in various food factories and households. Environmental samples from eight of nine food factories and from five of 16 households contained E sakazakii. The widespread nature of this micro-organism needs to be taken into account when designing preventive control measures.

A biofilm model for flowing systems in the food industry.

When bacteria attach to the walls of pipelines, they can form biofilms, which can cause the recontamination of food products. In order to quantify such recontamination, a one-dimensional biofilm model was developed taking into account adsorption, desorption, and the growth of cells. The model consisted of two mass balances describing increases in biofilm formation at the wall and the accumulation of cells in the liquid phase. The necessary parameters for the model were obtained in laboratory biofilm experiments. These experiments involved a flowing system and the use of Staphylococcus aureus as a model pathogen and silicon tubing as a testing material. S. aureus was inoculated into the system for 2 h, and then the system was changed to a sterile medium. Both biofilm formation and the release of cells into the flowing liquid were measured until steady-state conditions were reached (for up to 9 days). The experiments were performed in duplicate for different flow conditions (i.e., for Reynolds numbers of 3.2, 32, and 170). It was shown that at higher Reynolds numbers, the biofilm developed faster, probably owing to an increase in the transfer of nutrients to the surface. The proposed biofilm model was capable of describing the data obtained for the three different flow conditions with the use of the specific growth rate in the biofilm and the desorption coefficient as fit parameters. The specific growth rates were 0.16, 0.27, and 0.49 h(-1) for Reynolds numbers of 3.2, 32, and 170, respectively, and the desorption coefficients were about 1% of these values.

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants.

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.

Critical notes on microbiological risk assessment of food

Although numerous papers on Microbiological Risk Assessment (MRA) of food products have been published, a number of issues related to it remain unresolved. This paper explains the role of microbiological Risk Assessment in the context of Risk Analysis as outlined by Codex Alimentarius. It reviews some representative work in the area, with particular emphasis on the objectives, outputs and conclusions of the studies, and on how researchers propose using the resulting Risk Estimate for decision making. Several problems and sources of confusion concerning MRA are identified, such as terminology, the application of Risk Estimates to establish Food Safety Obejectives and microbiological criteria for foods, lack of data, and the difference between Risk Assesment and the Hazard Analysis and Critical Control Point system (HACCP). In the context of Codex Alimentarius, MRA was developed as a tool for decision making and priority setting by government risk managers. However, elements of Risk Communication. Industrial food safety managers can compare the effect of various hypothetical production scenarios using estimates of the level and the probability of a pathogen in the product at the time that is consumed. This limited form of Risk Assessment could better be called Safety Assessment, and can be used as a tool product and process development.