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.

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.

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.

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.

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.

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.

[Reconsideration of the 'bacteriological examination' of slaughter stock]. / Herbezinning over het 'Bacteriologisch Onderzoek' van slachtdieren.

A comparative trial was made by six meat inspection services to attain uniform indications for the 'Bacteriological Examination' of slaughtered animals in accordance with the 'Meat Inspection Regulations' of the Dutch 'Meat Inspection Act'. The results of the study compelled the investigators to scrutinize these regulations. The 'Bacteriological Examination' which is prescribed, is designed to exclude septicaemic animals from human consumption. Applying the rules will result in condemnation of animals harbouring a small number of bacteria which have a minimum significance for the health of consumers, whereas animals harbouring organisms potentially pathogenic for man are overlooked. It is concluded that the 'Meat Inspection Regulations' should be considered.

[More than 10 years of kidney testing in the Netherlands, and now...?]. / Ruim 10 jaar Nederlandse Nier Test, en nu....?

A historical review of testing for antibacterial residues in slaughtered animals is presented. In recent developments, much attention was paid to microbiological methods. Because of changing views and new possibilities increasing attention is being paid to purely chemical methods of detection. However, so long as chemical multi-purpose methods are not available, there will be a need for microbiological methods. The common misunderstanding that prevention of residues of antibacterial compounds in slaughter animals will contribute to reduction of the problem of bacterial resistance to antibacterial agents, is warned against.

Reference material for the evaluation of a standard method for the detection of salmonellas in foods and feeding stuffs.

To evaluate a standard salmonella isolation method a reference material consisting of 0.2 g spray-dried milk inoculated with Salmonella typhimurium and contained in gelatin capsules was prepared. The organisms were distributed homogeneously between capsules, and their numbers were stable for 120 d when the capsules were stored in dry conditions at 4 degrees C. Addition of these capsules with or without food samples to pre-enrichment broth gave low and reproducible levels of Salm. typhimurium contamination without altering the pre-enrichment and without influencing the other bacterial flora present. As a result of an interlaboratory trial, the reference material indicated that the food and/or its competitive flora may have a negative influence on the detection of salmonellas.

Growth and enterotoxin production of Staphylococcus aureus in shrimp.

Strains of Staphylococcus aureus isolated from shrimp were examined for phage pattern and enterotoxin production; 63% of the strains isolated from North Sea shrimp were typable with the International and additional set of phages, as were 38% of the strains isolated from South-East Asian shrimp. Staphylococcal enterotoxin(s) (SE) were produced by 48% and 35% of strains isolated from North Sea and South-East Asian shrimp respectively. Growth and enterotoxin production by S. aureus in shrimp was examined in storage experiments at 22 degrees C. S. aureus increased by 1-2 log units in 24 h when the organism was only a minor part of the total microflora of shrimp. When S. aureus was an equivalent part of the total flora its numbers increased by 3-4 log units in 24 h. Enterotoxins A and B became detectable when the number of S. aureus exceeded 10(7) per g in aseptically peeled shrimp. Results indicate that S. aureus is able to produce enterotoxin in shrimp, but its production depends upon a number of factors, including the relationship between S. aureus and competitive micro-organisms. It is concluded that the presence of S. aureus on commercially produced shrimp represents a potential hazard to health.

Collaborative study on the isolation of salmonella from artificially contaminated milk powder.

The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4 degrees C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4 degrees C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure.

[Detection of antibiotics and/or chemotherapeutic agents in slaughtering pigs by a captive impedance measurement using the M 120 Bactometer]. / Het aantonen van antibiotica- en/of chemotherapeutica in slachtvarkens door middel van capacitieve impedantiemeting met een Bactometer M 120.

A study was performed to compare the use of capacitive impedance measurement with EEC 4-plate method for the detection of antibacterial residues. Urine, meat, kidney and spleen were the matrices used in the impedance measurement. Meat was used in the EEC 4-plate method. Capacitive impedance measurement is quick, relatively simple, and reliable method, in which objective interpretation of the test results is guaranteed by automatic apparatus. Using meat and kidney as matrices, the test appears to be an efficient screening test for export meat and edible offal. As far as domestic Meat Inspection legislation is concerned, the test described may provide more specific information than the methods presently in use.