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Microbial survival of heating and cross-contamination are the two transmission routes during food preparation in the consumers' kitchen that are relevant for QMRA (Quantitative Microbial Risk Assessment). The aim of the present study was to extend the limited amount of data on microbial survival during real-life preparation of meat and meat products and to obtain accessory temperature data that allow for a more general (product unspecific) approach. Therefore survival data were combined with extensive measurements of time- and location dependent temperature using an infrared camera for the surface and buttons for the inside of the product, supplemented with interpolation modelling. We investigated the survival of heating of Escherichia coli O111:H2 in beefsteak, hamburgers (beef and 50% beef 50% pork (HH)), meatballs (beef and HH) and crumbs (HH). For beefsteak, survival as a whole is dominated by the sides, giving a log reduction of 1-2 (rare), 3-4 (medium) and 6-7 (done). Limited measurements indicated that done preparation gave 5-6 log reduction for crumbs and at least 8-9 log for the other products. Medium preparation gave a higher reduction in hamburgers (2-4 log) than in meatballs (1-2 log) and in beef (3-4) than in HH (2-3) hamburgers. In general, our 'done' results give larger inactivation than found in literature, whereas 'rare' and 'medium' results are similar. The experiments resulted in two types of curves of D70/z-values, dependent on product, doneness and for beefsteaks sides vs. top/bottom. One type of curve agrees reasonably with literature D70/z estimates from isothermal temperature experiments, which supports using these estimates for home style cooking QMRA calculations. In case of the other type of curve, which is mainly found for (near) surface contamination in close contact with the pan, these literature estimates cannot be applied. We also applied a simplified approach, assuming thermal inactivation is dominated by the highest temperatures reached. The time duration of this highest temperature gives accessory D-values which prove to fit with isothermal temperature literature data, thus suggesting application of such data for QMRA is possible by this approach also, which is less labor intensive both in terms of measurements and modelling. In real life, variability in product properties and preparation styles is large. Further studies are needed to analyze the effect on survival, preferably focusing on determining the essential variables. More variation in heating time will allow for estimating D70/z point estimates rather than curves representing possible sets of D70/z-values.

A methodology is presented to optimize a sampling plan for retail products to monitor the prevalence of foodborne pathogens in relation to disease burden. The optimization procedure links an exposure assessment, a quantitative measure for disease burden (i.e. Disability Adjusted Life Years), number of samples to be analyzed (based on uncertainty of the prevalence estimate) and costs for sample analysis. The methodology attributes DALY's on ‘pathogen-matrix level’ to ‘pathogen-food products’ using the exposure assessment. The subsequent procedure includes the number of samples that need to be analyzed per retail product such that the prevalences can be monitored within a preset uncertainty bound, and the costs per sample. The final optimization step sorts pathogen-product combinations using the costs per DALY criterion which results in a monitoring program with a maximum number of DALY's given a certain amount of money. An optimized sampling plan was established for four foodborne pathogens on meat products: Campylobacter in pork and poultry meat, Salmonella in pork, Toxoplasma in pork and Shiga-toxin producing Escherichia coli (STEC) O157 in beef, veal, and mutton/lamb. Results show that Campylobacter on poultry products and one Toxoplasma – pork combination consitute the top 10 in the proposed Dutch public health risk meat monitoring program. This optimized sampling plan monitors 98% of the total amount of DALY's attributed to the considered pathogen-animal species combinations. At the same time, the procedure gives insight in how the preset optimization criteria leads to the proposed set of pathogen-product combinations. An iterative implementation of updated model input (prevalences, DALY estimates) will lead to an up-to-date optimized risk based monitoring program.

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 that 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 the two 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.

The potential for using whole genome sequencing (WGS) data in microbiological risk assessment (MRA) has been discussed on several occasions since the beginning of this century. Still, the proposed heuristic approaches have never been applied in a practical framework. This is due to the non-trivial problem of mapping microbial information consisting of thousands of loci onto a probabilistic scale for risks. The paradigm change for MRA involves translation of multidimensional microbial genotypic information to much reduced (integrated) phenotypic information and onwards to a single measure of human risk (i.e. probability of illness). In this paper a first approach in methodology development is described for the application of WGS data in MRA; this is supported by a practical example. That is, combining genetic data (single nucleotide polymorphisms; SNPs) for Shiga toxin-producing Escherichia coli (STEC) O157 with phenotypic data (in vitro adherence to epithelial cells as a proxy for virulence) leads to hazard identification in a Genome Wide Association Study (GWAS). This application revealed practical implications when using SNP data for MRA. These can be summarized by considering the following main issues: optimum sample size for valid inference on population level, correction for population structure, quantification and calibration of results, reproducibility of the analysis, links with epidemiological data, anchoring and integration of results into a systems biology approach for the translation of molecular studies to human health risk. Future developments in genetic data analysis for MRA should aim at resolving the mapping problem of processing genetic sequences to come to a quantitative description of risk. The development of a clustering scheme focusing on biologically relevant information of the microbe involved would be a useful approach in molecular data reduction for risk assessment.

The presence of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli (EEC) in food animals, especially broilers, has become a major public health concern. The aim of the present study was to quantify the EEC exposure of humans in The Netherlands through the consumption of meat from different food animals. Calculations were done with a simplified Quantitative Microbiological Risk Assessment (QMRA) model. The model took the effect of pre-retail processing, storage at the consumers home and preparation in the kitchen (cross-contamination and heating) on EEC numbers on/in the raw meat products into account. The contribution of beef products (78%) to the total EEC exposure of the Dutch population through the consumption of meat was much higher than for chicken (18%), pork (4.5%), veal (0.1%) and lamb (0%). After slaughter, chicken meat accounted for 97% of total EEC load on meat, but chicken meat experienced a relatively large effect of heating during food preparation. Exposure via consumption of filet americain (a minced beef product consumed raw) was predicted to be highest (61% of total EEC exposure), followed by chicken fillet (13%). It was estimated that only 18% of EEC exposure occurred via cross-contamination during preparation in the kitchen, which was the only route by which EEC survived for surface-contaminated products. Sensitivity analysis showed that model output is not sensitive for most parameters. However, EEC concentration on meat other than chicken meat was an important data gap. In conclusion, the model assessed that consumption of beef products led to a higher exposure to EEC than chicken products, although the prevalence of EEC on raw chicken meat was much higher than on beef. The (relative) risk of this exposure for public health is yet unknown given the lack of a modelling framework and of exposure studies for other potential transmission routes.

The species Bacillus cereus, known for its ability to cause food borne disease, consists of a large variety of strains. An important property for discrimination of strains is their growth temperature range. Psychrotrophic strains can grow well at refrigerator temperatures but grow at 37 degrees C with difficulty. Mesophilic strains on the other hand are unable to grow below 10 degrees C, but grow well at 37 degrees C. Spores of six psychrotrophic and six mesophilic strains were investigated for their ability to survive and grow in simulated gastro-intestinal fluids, mimicking the conditions in the gastro-intestinal tract. The germination potential of psychrotrophic and mesophilic spores in simulated intestinal fluid does not differ much. Under conditions simulating the gastro-intestinal passage, 5 out of 6 mesophilic strains showed growth, and only 2 out of 6 psychrotrophic strains. Temperature (37 degrees C) and simulated gastro-intestinal conditions together influenced germination and growth.

Recent outbreaks with vegetable or fruits as vehicles have raised interest in the characterization of the public health risk due to microbial contamination of these commodities. Because qualitative and quantitative data regarding prevalence and concentration of various microbes are lacking, we conducted a survey to estimate the prevalence and contamination level of raw produce and the resulting minimally processed packaged salads as sold in The Netherlands. A dedicated sampling plan accounted for the amount of processed produce in relation to the amount of products, laboratory capacity, and seasonal influences. Over 1,800 samples of produce and over 1,900 samples of ready-to-eat mixed salads were investigated for Salmonella enterica serovars, Campylobacter spp., Escherichia coli O157, and Listeria monocytogenes. The overall prevalence in raw produce varied between 0.11% for E. coli O157 and L. monocytogenes and 0.38% for Salmonella. Prevalence point estimates for specific produce/pathogen combinations ranged for Salmonella from 0.53% in iceberg lettuce to 5.1% in cucumber. For Campylobacter, this ranged from 0.83% in endive to 2.7% in oak tree lettuce. These data will be used to determine the public health risk posed by the consumption of ready-to-eat mixed salads in The Netherlands.

The objective of this study was to evaluate the microbial hazard associated with the consumption of mixed salads produced under standard conditions. The presence of Salmonella, Campylobacter spp., and Escherichia coli O157 in the Dutch production chain of mixed salads was determined. Microbial prevalence and concentration data from a microbiological surveillance study were used as inputs for the quantitative microbial risk assessment. Chain logistics, production figures, and consumption patterns were combined with the survey data for the risk assessment chain approach. The results of the sample analysis were used to track events from contamination through human illness. Wide 95% confidence intervals around the mean were found for estimated annual numbers of illnesses resulting from the consumption of mixed salads contaminated with Salmonella Typhimurium DT104 (0 to 10,300 cases), Campylobacter spp. (0 to 92,000 cases), or E. coli (0 to 800 cases). The main sources of uncertainty are the lack of decontamination data (i.e., produce washing during processing) and an appropriate dose-response relationship.

[This corrects the article DOI: 10.1371/journal.pone.0169589.].

From a quantitative microbial risk assessment perspective it is important to know whether certain food environments influence the pathogenic potential of pathogens and to what extent. The purpose of the present study was to examine the pathogenic potential of S. Typhimurium DT104, measured as the capability to survive a simulated gastrointestinal tract system and the capability of adhering to and invading differentiated Caco-2 cells, after sequential incubation (without intermediate culturing) into soil, lettuce and cut lettuce stored under modified atmosphere (MAP) conditions. Two S. Typhimurium DT104 strains were used, one isolated from a pig carcass and one isolated from lettuce. The most important result of the present study is that the sequential incubation of S. Typhimurium in soil and lettuce slightly increased the capability of surviving the simulated gastric fluid, increased the capability to grow in the simulated intestinal fluid but decreased the capability of epithelial attachment and invasion and decreased the overall survival probability of the gastrointestinal tract system. Some variation in responses between the strains was observed, with the lettuce strain maintaining higher epithelial attachment capability and the carcass strains maintaining higher epithelial invasion capability. This study provided quantitative data on the effect of environmental and food matrices on the pathogenic potential of S. Typhimurium DT104 using a realistic system of sequential incubations in environmental and food matrices, followed by simulated gastrointestinal tract passage without intermediate culturing. These results could aid the development of more realistic quantitative microbial risk assessments.

Spores obtained from Bacillus cereus ATCC 14579 and mutant strains lacking each of seven germinant receptor operons were exposed to differentiated Caco-2 cells and monitored for germination. Spores of the gerI and gerL mutants showed a reduced germination response, pointing to a role for these receptors in Caco-2-induced germination.

Spores of 11 enterotoxigenic strains of Bacillus cereus isolated from foods and humans adhered with similar efficiencies to Caco-2 cells, whereas subsequent germination triggering was observed with only 8 of these strains. Notably, Hep-2 cells did not trigger germination, while spores of all strains displayed similar germination efficiencies in brain heart infusion broth.

Randomly selected food commodities, categorized in product groups, were investigated for the presence and number of Bacillus cereus bacteria. If positive, and when possible, five separate colonies were isolated and investigated for the presence of four virulence factors: presence of genes encoding three enterotoxins (hemolysin BL [HBL], nonhemolytic enterotoxin [NHE], and cytotoxin K) and the ability to produce cereulide. In addition, the presence of psychrotrophic and mesophilic signatures was determined. The genes for NHE are found in more than 97% of the isolates, those for HBL in approximately 66% of the isolates, and the gene for cytotoxin K in nearly 50% of the isolates. Significant associations between product groups and (combinations of) virulence factors were the relatively low percentage of isolates from the "flavorings" group containing genes encoding NHE and the higher-than-average occurrence of both the genes encoding HBL and NHE in the "pastry" group. Cereulide was produced by 8.2% of the isolates but only in combination with the presence of genes for one or more other virulence factors. Most isolates (89.9%) were mesophilic; minorities of the isolates were psychrotrophic (4.4%) or of intermediate signature (5.7%). In the product group "milk and milk products," the incidence of strains with psychrotrophic or intermediate signatures is significantly higher than in the other product groups. In the product groups "flavorings," "milk and milk products," "vegetable(s) and vegetable products," "pastry," and "ready-to-eat foods," a relatively high number of samples contain high numbers of B. cereus bacteria. Within the product group "ready-to-eat foods," the products containing rice and pasta show a relatively high incidence of high numbers of B. cereus bacteria.