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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.

Bacterial microcompartment (BMC) dependent ethanolamine (eut) and propanediol utilization (pdu) has recently been shown to stimulate anaerobic growth of Listeria monocytogenes. This metabolic repertoire conceivably contributes to the competitive fitness of L. monocytogenes in the human gastrointestinal (GI) tract, where these compounds become available following phospholipid degradation and mucus-derived rhamnose metabolism by commensal microbiota. Previous transcriptomics and mutant studies of eut and pdu L. monocytogenes suggested a possible role of eut and pdu BMC metabolism in transmission in foods and pathogenicity, but data on a potential role of L. monocytogenes interaction with human cells is currently absent. First, we ask which cellular systems are expressed in the activation of eut and pdu BMC metabolism and the extent to which these systems are conserved between the states. We find common and unique systems related to metabolic shifts, stress and virulence factors. Next, we hypothesize that these common and unique activated cellular systems contribute to a role in the interaction of L. monocytogenes interaction with human cells. We present evidence that metabolically primed L. monocytogenes with active eut and pdu BMCs, as confirmed by metabolic analysis, transmission electron microscopy and proteomics, show significantly enhanced translocation efficacy compared to non-induced cells in a trans-well assay using Caco-2 cells, while adhesion and invasion capacity was similar. Taken together, our results provide insights into the possible key cellular players that drive translocation efficacy upon eut and pdu BMC activation.

Listeria monocytogenesis a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded byL. monocytogenesin aerobic and anaerobic conditions into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulates anaerobic growth ofL. monocytogeneson rhamnose due to the activation of bacterial microcompartment (BMC)-dependent 1,2-propanediol utilization with concomitant production of propionate and propanol. Notably, anaerobic propanediol metabolism has been linked to virulence of enteric pathogens includingSalmonellaspp. andL. monocytogenes. In this study we investigate the impact of B12 on aerobic and anerobic growth ofL. monocytogeneson rhamnose, and observed growth stimulation andpduBMC activation only in anaerobically grown cells with B12 added to the medium. Comparative Caco-2 virulence assays, showed that thesepduBMC induced cells have significantly higher translocation efficiency compared to aerobically grown cells (without and with added B12) and non-induced anaerobically grown cells, while adhesion and invasion capacity is similar for all cells. Comparative proteomics analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL; teichoic acids export ATP-binding protein, TagH; DNA repair and protection proteins RadA and DPS; and glutathione synthase GshAB previously linked to activation of virulence response inL. monocytogenes, uniquely upregulated in anaerobically rhamnose grownpduBMC induced cells. Our results shed new light into B12 impact onL. monocytogenescompetitive fitness and virulence.

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 behavior of 35 different Salmonella enterica serovars was investigated in an in vitro gastro-intestinal tract (GIT) system. Virulence was expressed as the probability of infection, P(inf), i.e. fraction of the ON-culture invading into Caco-2 cells after GIT passage. Results show that the (average) P(inf) of Salmonella serovars ranges from 1.7 10-8 (S.Kedougou) to 5.3 10-5 (S.Typhimurium). In general, the P(inf) corresponds to available epidemiological and virulotypic data from literature. Still, individual exceptions exist and it is hypothesized that the public health risk from Salmonella is associated with exposure (prevalence, dose and/or acquired immunity) rather than difference in virulence.

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.

Background European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. Methods Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. Results Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. Conclusions European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.

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.

The estimation of exposure to molds and their products in the indoor environment, which may lead to the occurrence of allergies or respiratory complaints, by means of enumeration of viable parts is inadequate. Therefore, other methods must be developed. When grown under various circumstances (22°C and 30°C, high and low water activity) under laboratory conditions, Alternaria alternata produces one antigen that can be found under all studied growth conditions in extracts of the water-soluble portion of the mycelium. This common antigen may serve as marker antigen for exposure to A. alternata and its allergens. In extracts of the culture filtrate, three antigens, designated index antigens, have been identified that together may have the function of marker for the exposure to allergens of A. alternata.

[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.

Aims: Model the number of viable vegetative cells of B. cereus surviving the gastric passage after experiments in simulated gastric conditions. Materials and Methods: The inactivation of stationary and exponential phase vegetative cells of twelve different strains of Bacillus cereus, both mesophilic and psychrotrophic strains isolated from food and faeces from healthy and ill individuals, in simulated gastric conditions was determined using decimal reduction times at low pH (DpH). Subsequently inactivation rates were calculated. Inclusion of the inactivation rates into models describing the course of the gastric pH after the consumption of meal of solid food and the transfer of food from the stomach to the small intestine resulted in numbers of viable Bacillus cereus vegetative cells able to pass the stomach. Conclusions: According to the model, 3¿26% of the ingested vegetative cells from Bacillus cereus may survive the gastric passage, dependent on the growth phase of the vegetative cells, the type of strains, and the age of the consumer. Significance and Impact of the Study: Vegetative cells of Bacillus cereus may be involved in the onset of diarrhoeal disease to a greater extent than expected since up to 26% of the ingested cells survive simulated gastric conditions.

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.

Serial urine samples were collected from 33 neutropenic patients, 10 of whom developed invasive aspergillosis (IA) while undergoing bone marrow transplantation or remission induction therapy for leukemia. Concentrated urine samples from the infected patients were subjected to polyacrylamide gel electrophoresis, blotted, and then incubated with antiserum raised to a cell wall extract of Aspergillus fumigatus (anti-CW) or an immunoglobulin G monoclonal antibody to A. fumigatus galactomannan (EBA1). When IA patient urine blots were probed with anti-CW, major bands at 11 and 18 kilodaltons (kDa); intermediate bands at 13, 14, and 29 kDa; and minor bands at 38 and 44 kDa were seen. In contrast, EBA1 showed diffuse staining at molecular masses larger than 45 kDa and a single weak band at 21 kDa. Urine samples from the 23 patients with no evidence of IA were unreactive with both anti-CW and EBA1. These antigen bands are likely to represent immunodominant antigens which are excreted during IA and should play a valuable role in the development of rapid diagnostic tests for aspergillosis.

The presence of antibodies to extracellular polysaccharides (EPS) of moulds in sera of healthy subjects (N = 125) was determined. Antibodies against the EPS of Penicillium digitatum, Mucor racemosus, Cladosporium cladosporioides, Fusarium moniliforme and Botrytis tulipae were found in relatively high amounts in all sera. No effect of age on antibodies present could be demonstrated. Antibodies against each of the EPS tested were only neutralized by the homologous EPS and by EPS of moulds belonging to the same genus or a taxonomically closely related genus. Antibodies against the EPS of P. digitatum were inhibited by methyl-beta-D-galactofuranoside, indicating that the galactofuranose part of this EPS is immunodominant.