A QMRA Model for Salmonella in Pork Products During Preparation and Consumption.

As part of a quantitative microbiological risk assessment (QMRA) food chain model, this article describes a model for the consumer phase for Salmonella-contaminated pork products. Three pork products were chosen as a proxy for the entire pork product spectrum: pork cuts, minced meat patties, and fermented sausages. For pork cuts cross-contamination is considered the most important process and therefore it is modeled in detail. For minced meat, both cross-contamination and undercooking are the relevant processes. For those commodities bacterial growth during transport and storage is also modeled. Fermented sausages are eaten raw and the production may be defective. Variability between consumers' behavior and the impact of variability between production processes at the farm and abattoir are taken into account. Results indicate that Salmonella levels on products may increase significantly during transport and storage. Heating is very efficient at lowering concentrations, yet cross-contamination plays an important role in products that remain contaminated. For fermented sausage it is found that drying is important for Salmonella reduction. Sensitivity analysis revealed that cross- contamination factors "knife cleaning" and "preparation of a salad" are important parameters for pork cuts. For minced meat cleaning of the board, salad consumption, refrigerator temperature, and storage time were significant.

Consumption of raw vegetables and fruits: a risk factor for Campylobacter infections.

The purpose of this study was to determine the prevalence of Campylobacter in fresh vegetables and fruits at retail level in the Netherlands, and to estimate its implications on the importance of vegetables and fruits as risk factor for campylobacteriosis. Thirteen of the 5640 vegetable and fruit samples were Campylobacter positive, resulting in a prevalence of 0.23% (95% confidence interval (Cl): 0.12-0.39%). The prevalence of packaged products (0.36%, 95% Cl: 0.17-0.66) was significantly higher than of unpackaged products (0.07; 95% Cl: 0.01-0.27). No statistical differences were found between seasons. Combining the mean prevalence found in this study with data on the consumption of vegetables and fruits, an exposure of 0.0048 campylobacters ingested per person per day in the Netherlands by transmission via vegetables and fruits, was calculated. This exposure, as input in a Beta-Poisson dose-response model, resulted in an estimated number of 5.3×105 cases of infection with Campylobacter per year for the whole Dutch population. This constitutes the consumption of raw vegetables and fruits, especially when packaged, to be a risk factor for Campylobacter infections.

Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach.

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 (D(pH)). 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.

Quantification of Campylobacter jejuni cross-contamination via hands, cutlery, and cutting board during preparation of a chicken fruit salad.

Using artificially contaminated chicken, the quantitative overall effect of Campylobacter jejuni cross-contamination, either via cutlery, cutting board, or hands, on the microbiological quality of a chicken salad was tested to identify the most critical transfer route. The end contamination level of salads prepared according to different scenarios, with or without cross-contamination, was compared. It was shown that the mean transfer rate calculated for all salads prepared allowing cross-contamination was 0.12% of the initial number of C. jejuni on the chicken fillet (8.8 +/- 0.2 log CFU). The difference in calculated transfer rates for the tested cross-contamination routes was not significantly different (P > 0.05). The prevention of cross-contamination by replacing cutlery and cutting board after handling raw chicken and the prevention of hand contact resulted in considerably reduced end contamination levels (< 2.4 log CFU) or noncontaminated end products. The results of this study emphasize the importance of preventing cross-contamination during food handling in reducing the risks of foodborne infections, and they provide useful data for quantitative microbiological risk assessment.

Modelling of Campylobacter survival in frozen chicken meat.

AIMS: To model the survival kinetics of Campylobacter jejuni on frozen chicken meat. METHODS AND RESULTS: Three different types of chicken meat surface (skin, skinned muscle and cut muscle) were inoculated with stationary phase cells of C. jejuni (8 log(10) CFU cm(-2)) and frozen for 5 weeks at -20 degrees C. Bacterial numbers were determined weekly using two different methods of enumeration to quantify uninjured and injured cells. Analysis of variance of the results showed that the type of chicken surface and the method used to enumerate surviving cells were the most significant sources of variations in the numbers recovered (P < 0.0001), much more than the freezing time. To identify an appropriate model for the description of effects of freezing on survival over time, several models were fitted to the count data. Decay was found to be nonlinear. In general, survival was least on skin, better on skinned muscle and best on cut muscle. After 2 weeks, additional inactivation by freezing appeared to be negligible. CONCLUSION: Because of the variability of survival it was not possible to fit and select a general model useful for all the different surfaces types. SIGNIFICANCE AND IMPACT OF THE STUDY: The injured state of the cells leads to variability and the underestimation of bacterial survival. This is an essential factor for the assessment of Campylobacter-associated risk.

Germination of Bacillus cereus spores is induced by germinants from differentiated Caco-2 Cells, a human cell line mimicking the epithelial cells of the small intestine.

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.

Prevalence of potentially pathogenic Bacillus cereus in food commodities in The Netherlands.

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.

Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains.

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.

A one-year intensified study of outbreaks of gastroenteritis in The Netherlands.

In 2002, in The Netherlands a national study of gastroenteritis outbreaks was performed. Epidemiological information was collected by the Public Health Services (PHS) and the Food Inspection Services (FIS) using standardized questionnaires. Stool samples were collected for diagnostic testing. For foodborne outbreaks, food samples were taken. In total, 281 gastroenteritis outbreaks were included, mainly from nursing homes and homes for the elderly (57%), restaurants (11%), hospitals (9%) and day-care centres (7%). Direct person-to-person spread was the predominant transmission route in all settings (overall 78%), except for restaurant outbreaks where food was suspected in almost 90% (overall in 21% of outbreaks). The most common pathogen was norovirus (54%), followed by Salmonella spp. (4%), rotavirus group A (2%), Campylobacter spp. (1%) and only incidentally others. In conclusion, most outbreaks were reported from health and residential institutions, with norovirus as the dominant agent. Control should aim at reducing person-to-person spread. In foodborne outbreaks norovirus was common, due to contamination of food by food handlers. Salmonella, as the second foodborne pathogen, was mainly associated with raw shell eggs. These results stress the continuous need for food safety education, complementary to governmental regulation.

The adaptive response of Escherichia coli O157 in an environment with changing pH.

AIMS: To predict and validate survival of non-acid adapted Escherichia coli O157 in an environment mimicking the human stomach. METHODS AND RESULTS: Survival was predicted mathematically from inactivation rates at various, but constant pH values. Predictions were subsequently validated experimentally in a pH-controlled fermentor. Contrary to prediction, acid-sensitive cultures of E. coli O157 survived for a long period of time and died as rapidly as acid-resistant cultures. Experimental results showed that in an environment with changing pH, acid-sensitive cultures became acid-resistant within 17 min. Cyclo fatty acids was reported to be a factor in acid resistance. As synthesis of cyclo fatty acids does not require de novo enzyme synthesis and thus requires little time to develop, we analysed the membrane fatty acid composition of E. coli O157 during adaptation. No changes in membrane fatty acid composition were observed. CONCLUSIONS: Acid adaptation of E. coli O157 can occur during passage of the human gastric acid barrier, which can take up to 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of acid-adapted bacteria to survive the human stomach is an important virulence factor. The ability of non-acid adapted E. coli O157 to adapt within a very short period of time under extreme conditions further contributes to the virulence of E. coli O157.

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments.

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.

Sensor, a population-based cohort study on gastroenteritis in the Netherlands: incidence and etiology.

A prospective population-based cohort study with a nested case-control study was conducted to estimate the incidence of gastroenteritis and the associated pathogens in the general Dutch population. Follow-up of two consecutive cohorts was performed by weekly reporting cards from December 1998 to December 1999. Cases and controls in the case-control study supplied a questionnaire and stool samples. The standardized gastroenteritis incidence was 283 per 1,000 person-years. The incidence rose with increasing level of education and was higher for persons with a history of diarrhea and for young children. Bacterial pathogens accounted for 5% of cases, bacterial toxins for 9%, parasites for 6%, and viral pathogens for 21%, with Norwalk-like virus (NLV) as the leading pathogen in 11% of cases. The gastroenteritis incidence was higher than that reported for England, but lower than for the United States. In community cases, viral pathogens are the leading cause of gastroenteritis, with NLV being the number one cause of illness in all age groups but one. In many countries, preventive measures are implemented to decrease bacterial infections. However, additional prevention of viral infections, especially NLV, might significantly decrease the number of gastroenteritis cases in the community.

A rat model for dose-response relationships of Salmonella Enteritidis infection.

AIMS: To develop an animal model to study dose-response relationships of enteropathogenic bacteria. METHODS AND RESULTS: Adult, male Wistar Unilever rats were exposed orally to different doses of Salmonella enterica serovar Enteritidis after overnight starvation and neutralization of gastric acid by sodium bicarbonate. The spleen was the most sensitive and reproducible organ for detection of dose-dependent systemic infection. Illness was only observed in animals exposed to doses of 10(8) cfu or more. At lower doses, histopathological changes in the gastro-intestinal tract were observed, but these were not accompanied by illness. Marked changes in numbers and types of white blood cells, as well as delayed-type hyperresponsiveness, indicated a strong, dose-dependent cellular immune response to Salm. Enteritidis. CONCLUSION: The rat model is a sensitive and reproducible tool for studying the effects of oral exposure to Salm. Enteritidis over a wide dose range. SIGNIFICANCE AND IMPACT OF THE STUDY: The rat model allows controlled quantification of different factors related to the host, pathogen and food matrix on initial stages of infection by food-borne bacterial pathogens.

Quantification of the contamination of chicken and chicken products in the netherlands with Salmonella and Campylobacter.

The research described in this contribution provides quantitative data on contamination levels with Salmonella and Campylobacter in chicken and chicken products in The Netherlands at retail level using the most probable number method and direct counting. Most samples contained <10 Salmonella per carcass, both in fresh (89%) and frozen (68%) products, contamination levels with Campylobacter varied from <10 (18%) to more than 5,500 (18%) per fresh carcass. Most frozen samples (57%) contained < 10 Campylobacter per carcass.

Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassays of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk.

The presence of genes for the production of the three components of the HBL enterotoxin complex and enterotoxin-T in Bacillus cereus was evaluated by PCR tests for strains isolated from milk. In addition enterotoxin production of B. cereus was evaluated by means of the HBL blood agar plate and two commercially available toxin tests. All three genes for the HBL enterotoxin complex were detected in 55% of the 86 strains tested, the enterotoxin-T gene was detected in 62% of the strains. A few strains showed a weak reaction in the PCR tests for the L1 or L2 components of the HBL enterotoxin complex. Many strains that were found to contain the genes for the HBL complex gave negative or doubtful results in the HBL blood agar plate test. All strains that contain the L2 part of the HBL complex showed a titer of at least 8 in the Oxoid RPLA test. Two strains that did not contain the L2 part of the HBL enterotoxin complex gave high titers (= 64) in the RPLA test.

Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project.

Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation--from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment--consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards.

Marker antigens to assess exposure to molds and their allergens. II. Alternaria alternata.

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 degrees C and 30 degrees 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.

Marker antigens to assess exposure to molds and their allergens. I. Aspergillus fumigatus.

BACKGROUND: Exposure to molds and mold products in the indoor environment may lead to allergies, asthma, or respiratory complaints in general. Enumeration of viable parts of molds in the environment is insufficient to estimate exposure. Therefore, other methods have to be developed. METHODS: Aspergillus fumigatus (Af) was grown under various circumstances (22 degrees C and 30 degrees C, high and low water activity) in the laboratory. At various moments during culture, extracts were taken, and antigen and allergen content was examined by acrylamide electrophoresis and immunoblot. RESULTS: In extracts of the culture filtrate, two antigens were found to be produced under all studied growth conditions (common antigens). In the extracts of the water-soluble portion of the mycelium, one common antigen was found. CONCLUSIONS: The three common antigens may serve as marker antigens for exposure to Af and its products. In view of the simultaneous presence of two of these common antigens with Af allergens, these two marker antigens may be used to estimate exposure to allergens of Af.

Immunoblotting technique for the detection of allergens of Aspergillus fumigatus: influence of Nonidet P-40 on the sensitivity.

Immunoblotting provides a useful technique for the study of antigens, antibodies and allergens. To overcome problems regarding the loss of antigenic properties during the blotting and developing procedures, several solutions have been described. The inclusion of Nonidet P-40, recommended to increase the sensitivity of developing procedures for immunoblots, in an existing procedure for the detection of allergens of Aspergillus fumigatus, however, led to decreased sensitivity of the method.

Fungal extracellular polysaccharides in house dust as a marker for exposure to fungi: relations with culturable fungi, reported home dampness, and respiratory symptoms.

BACKGROUND: Epidemiologic studies have demonstrated an association between indoor fungal growth and respiratory symptoms. However, in only a few studies was fungal exposure actually measured. OBJECTIVE: The purpose of this study was to evaluate the measurement by enzyme immunoassay of extracellular polysaccharides of Aspergillus and Penicillium species (EPS-Asp/Pen ) in house dust as a marker for fungal exposure and to study the relations between EPS-Asp/Pen levels and home dampness and respiratory symptoms in children. METHODS: Extracts of house dust samples from bedroom and living room floors and mattresses from homes of 31 children with chronic respiratory symptoms and 29 children with no chronic respiratory symptoms were analyzed for EPS-Asp/Pen. RESULTS: EPS-Asp/Pen were readily detectable (40 to 46,513 nanogram equivalent/g dust) in 161 house dust extracts, with highest concentrations in living room floor dust. EPS-Asp/Pen levels were 2 to 3 times higher on carpeted floors than on smooth floors. EPS-Asp/Pen were significantly correlated with total culturable fungi (r = 0.3 to 0.5) and with house dust mite allergens (r = 0.3 to 0.5). EPS-Asp/Pen levels in living room floor dust were positively associated with occupant-reported home dampness. This was not observed for EPS-Asp/Pen in bedroom floor and mattress dust. EPS-Asp/Pen levels in living room floor dust were positively associated with respiratory symptoms. EPS-Asp/Pen in bedroom floor and mattress dust showed a reversed association with respiratory symptoms, possibly because of allergen-avoidance measures taken in the bedroom. CONCLUSION: The enzyme immunoassay for fungal EPS-Asp/Pen may be a useful method for exposure assessment of indoor fungi.