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.

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.

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.

Validation by an interlaboratory collaborative trial of EN ISO 21528 - microbiology of the food chain - horizontal methods for the detection and enumeration of Enterobacteriaceae.

The methods for the detection and enumeration of Enterobacteriaceae, described in EN ISO 21528, parts 1 and 2, were validated by order of the European Commission under the mandate M/381. Fourteen laboratories from seven European countries participated in the collaborative trials, organized by The Netherlands Food and Consumer Product Safety Authority (Wageningen/Utrecht, The Netherlands). Five different matrices from different food categories were selected to be tested in the collaborative trials, in order to validate the method horizontally, according to ISO 16140. The matrices included meat, tiramisu, infant formula, liquid egg, smoked salmon (detection method only) and animal feed (enumeration method only). The raw meat and liquid egg were naturally contaminated and the other matrices were artificially contaminated with a cocktail of four different Enterobacteriaceae strains. The samples used in the trial were tested for homogeneity and stability before distribution. The method for detection of Enterobacteriaceae showed a specificity and sensitivity above 95% for all matrices. The method for the enumeration had a repeatability limit r of 0.37 (expressed as a difference between log10-transformed test results) and a reproducibility limit R of 0.87 (expressed as a difference between log10-transformed test results). The validation data were incorporated in the newly published ISO standards EN ISO 21528:2017- Microbiology of the food chain - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 1: Detection method, and Part 2: Colony-count technique.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method.

During the last decade Campylobacter has been the most commonly reported gastrointestinal bacterial infection in humans in the European Union. The use of a sensitive detection method based on enrichment of Campylobacter spp. is often needed when examining foods. However, as background flora developed resistance to third generation ß-lactams used in selective culture media, the ISO method was adapted. It now consists of three different procedures (A, B, and C) depending on the expected concentration and condition of Campylobacter and the background microflora. As the diagnostic sensitivity of the detection test varies between laboratories, this justifies the validation of the method in an interlaboratory study. The matrices selected for testing in the collaborative trials were frozen spinach (procedure A, Bolton enrichment broth), minced meat (procedure A, Bolton enrichment broth), raw milk (procedure B, Preston enrichment broth), chicken skin (procedure B, Preston enrichment broth), and broiler caecal material (procedure C, direct plating on mCCD agar). Each matrix was artificially inoculated with a different Campylobacter strain at a low and high contamination level, and with sterile diluent for 'blanks'. Seventeen laboratories participated in the interlaboratory study. The sensitivity and specificity of the methods for the five selected matrices were determined, as well as the level of detection (LOD50). Calculated LOD50 values ranged from 0.84 cfu/test portion in frozen spinach and 2.2 cfu/test portion in minced meat to 14 cfu/test portion in chicken skin and 57 cfu/test portion in raw milk, all based on test portions of 10 g. The test portion size for broiler caecal material was a 10 µl-loop, yielding a LOD50 of 6.1 cfu/test portion. The validation data were incorporated in the newly published ISO standard EN ISO 10272-1:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 1: Detection method.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique.

The validation in an interlaboratory study of the International Standards Organization standard method for the enumeration of Campylobacter in foods (ISO 10272-2) was performed after preparation of the revised Standard based on scientifically sound and validated methods of analysis. The matrices selected for testing in the collaborative trial were frozen spinach, minced meat, raw milk, chicken skin, and broiler caecal material. Each matrix was artificially inoculated with a different Campylobacter strain. Fifteen laboratories participated in the interlaboratory study. As a general indication of repeatability limit (r), the following overall values can be used when testing chicken skin samples: As a general indication of reproducibility limit (R), the following overall values can be used when testing chicken skin samples: The validation data for all matrices were incorporated in the newly published ISO standard EN ISO 10272-2:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 2: colony-count technique.

Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR.

A multiplex quantitative PCR (qPCR) was developed and evaluated for the simultaneous detection of Salmonella spp., S. enterica serovar Typhimurium and S. enterica serovar Enteritidis in various (food) matrices. Early and fast detection of these pathogens facilitates effective intervention and prevents further distribution of contaminated food products on the market. Three primer and probe sets were designed to target the invA gene, the STM4200 gene, and the SEN1392 gene to detect and differentiate Salmonella spp., S. Typhimurium, and S. Enteritidis, respectively. The multiplex qPCR targeting these three genes was optimized for efficiency and linearity. By testing 225 Salmonella isolates and 34 non-Salmonella isolates from various sources the inclusivity and exclusivity were determined. The inclusivity of the multiplex qPCR was 100% for all Salmonella isolates, including 72 S. Typhimurium isolates, and 53 S. Enteritidis isolates. The exclusivity for Salmonella spp., S. Typhimurium, and S. Enteritidis was 100%, 94.6%, and 100%, respectively. No positive results were reported for non-Salmonella isolates. The limit of detection (LOD) for the qPCR was determined for the matrices poultry, minced meat, egg, herbs/spices, powdered milk, fish, animal feed, boot-socks with chicken feces and chicken down. LOD values for qPCR and the conventional culture methods were similar, except for the matrix boot-socks and down, for which the LOD for the conventional culture methods performed better than the qPCR method. In conclusion, the multiplex qPCR assay developed allows for rapid screening of Salmonella spp., S. Typhimurium, and S. Enteritidis in various (food) matrices.

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.

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.