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.

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.

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.

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.

[Incidence of Clostridium botulinum on cattle farms]. / Voorkomen van Clostridium botulinum op rundveehouderij bedrijven.

Clostridium botulinum, mainly type B, was constantly found to be present on cattle farms. The organism was isolated both from samples of the soil of pastures and from the faeces of cattle during the winter housing period. The number of C. botulinum type B in samples of soil varied from 10 to 300 organisms per 100 grams. Contamination with C. botulinum was found to be of a similar order of magnitude on farms on which pastures are regularly dressed with sewage sludge. C. botulinum was detected in 13 per cent of the faecal samples (420 samples of one gram each). Particularly grass silage pits prepared with wilted grass were found to provide a possible link between contamination of pastures and cattle.

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.

A simple purification method for enterotoxin F produced by Staphylococcus aureus and some properties of the toxin.

Staphyloccoccus aureus enterotoxin F (SEF), which is associated with S. aureus strains isolated from toxic-shock-syndrome patients, was purified by successive chromatography on CM sephadex C-25 and gelfiltration on sephadex G-75. When tested by disc-polyacrylamide gel-electrophoresis the toxin migrated as a homogeneous protein. In SDS-polyacrylamide gel-electrophoresis three protein bands were observed. The main component had a mol wt of 23 000 and the two minor components had a mol wt less than 13 000. By iso-electric focussing a main protein band with an iso-electric point of 7.2 was obtained. The LD50 for rabbits (3-3.5 kg) by subcutaneous and intravenous application of SEF was 6 micrograms and 180 micrograms, respectively. Antibodies to SEF prepared in a sheep did not react with other staphylococcal enterotoxins (A to E).

The identification of Salmonella enteritidis-infected poultry flocks associated with an outbreak of human salmonellosis.

In the summer of 1991 a human outbreak of Salmonella enteritidis infection occurred following a barbecue in which about 100 persons were involved. Eggs, supplied by one or more of 10 different layer farms, were the most probable source of the infection. To identify the S. enteritidis-positive flocks, an immunoassay was used to detect salmonella serogroup D-specific antibodies in the yolk of hens eggs. Antibody titres in the eggs from two layer farms, farm A and B, clearly exceeded the titres found in randomly collected eggs. Further investigation on farm A and B yielded high antibody titres in the eggs from flocks A1, A2 and B2, and low titres in the eggs from flock B1. S. enteritidis was isolated from the faecal samples of flocks A1, A2 and B2, whereas no salmonella was detected in the faecal samples of flock B1. The flocks present on both farms originated from the same breeder flock.

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.