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.

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 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.

Studies on the risk assessment of Listeria monocytogenes.

Humans are frequently exposed to Listeria monocytogenes, and high numbers may be ingested during consumption of certain types of food. However, epidemiological investigations show that listeriosis is a rare disease. Risk assessment studies using an animal mouse model indicate that almost all L. monocytogenes serovars present in food have clear virulent properties. The intravenous dose causing infection in 50% (IV ID50) of mice not previously exposed to L. monocytogenes (nonprotected mice) was 1.8 log(10) units. For mice previously exposed to L.monocytogenes (immunologically protected mice was >9.0 log10 5.6 log(10) units. The ID(50) of orally exposed nonprotected mice amounted to 6.5 log10 units, and no significant effects of type of food (water/milk) and storage time at 5 degrees C (milk) were observed. The oral ID50 of immunologically protected mice was >9.0 log10 units. Furthermore, there was approximately 1-2 log10 difference between the ID50 and the lethal dose causing death in 50% (LD50). The results show that both the intestinal barrier and the specific immune defense mechanism are highly effective in preventing infection of mice orally exposed to L.monocytogenes. Delaying the immune defense had no effect on the protective activity of the intestinal barrier, indicating that these protective mechanisms independently. The risk assessment results obtained in the mouse model support the epidemiological finding that listeriosis is a rare disease in humans, despite frequent exposure to the organism.

The effect of previous growth conditions on the lag phase time of some foodborne pathogenic micro-organisms.

In current models for predicting microbial growth, the lag phase duration is expressed as a function of the growth rate of the micro-organism. We observed that in addition to the growth rate (as influenced by incubation temperature and NaCl contents), the pre-incubation temperature influences the lag phase duration of foodborne pathogenic micro-organisms.

Characteristics of some psychrotrophic Bacillus cereus isolates.

Twelve strains of Bacillus cereus isolated from different food products and foodborne disease outbreaks, and able to grow at temperatures < 7 degrees C, were characterised. Generation times at 7 degrees C varied from 9.4 h up to 75 h. Lag phase of the vegetative cells at 7 degrees C was strongly influenced by the previous temperature history of the cells. Preincubation at 37 degrees C increased the duration of the lag phase drastically. The heat resistance at 90 degrees C (D90 degrees C-values in min) for spores produced at 30 degrees C varied from 2.2 to 9.2 min for 11 strains. One strain, however, showed a D90 degrees C-value of > 100 min. Germination of spores in milk was delayed compared to those grown in brain heart infusion broth (BHI). All strains showed production of the diarrheal type enterotoxin in BHI. Addition of 50 IU of nisin to skim milk resulted in a decrease of numbers for 9 of the 12 strains tested. At a nisin concentration of 250 IU, a decrease in bacterial numbers was observed for all strains tested.

Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes.

Experimental infections of mice with strains of Listeria spp. isolated from contaminated food sources allowed discrimination of strains into those either exhibiting high, attenuated or low virulence. Compared to the highly virulent L. monocytogenes strain EGD, an attenuated strain such as L99 persisted for shorter times (5 versus 10 days) in the infected host. Using a tissue culture cell model of infection, we found that, although strain L99 was capable of accumulating actin like its virulent counterpart following invasion, it was unable to generate the polarized actin tails required for intracellular and cell-to-cell movement. Immunoblot analysis using specific antiserum to the ActA polypeptide, a molecule that is necessary for movement of the bacterium within the eucaryotic cell, indicated that a slightly truncated form of this polypeptide was produced in the L99 strain. Despite its reduced virulence, the attenuated strain L99 was just as effective in generating protection in immune mice as the highly virulent strains, albeit with a 1000-fold higher infective dose. Based on the results obtained from this study, we suggest that one of the mechanisms accounting for widespread resistance in humans to infection by Listeria may be due to asymptomatic infections by naturally occurring strains attenuated for virulence.

Stability of spores of Bacillus cereus stored on silicagel.

Spores of four different strains of Bacillus cereus were stored on silicagel at 22 degrees C and in physiological saline solution at -20 degrees C for a period of 260 days. At different intervals the spores were tested for survival, heat sensitivity and capacity to germinate. There was no clear change in any of the parameters tested, so storage on silicagel can be a good alternative for storage as a frozen suspension. Spores stored in this way can easily be exchanged for reference material and used for microbiological challenge testing.

Characteristics of Bacillus cereus related to safe food production.

Thirty Bacillus cereus strains, isolated from different sources, were characterized in relation to safe food production. The minimal growth temperatures of the strains varied from < or = 5 degrees C to 11 degrees C. Generation times at 7 degrees C of strains capable of growing at temperatures < or = 5 degrees C were approximately 8.2 h. The D90 degrees C-values of spores of strains with a minimal growth temperature of 11 degrees C determined in phosphate buffer at pH 7.0 ranged from 4.8 to > 200 min. Strains with the capacity to grow at temperatures < or = 9 degrees C, had a D90 degrees C value ranging from 4.6 to 14 min. Addition of either nisin (250 micrograms/ml) or diacetyl (1500 micrograms/ml) to the heating menstruem at the single concentrations investigated seemed not influence the thermal destruction of spores. Germination of spores of almost all strains occurred in all three media tested (Brain Heart Infusion, rice extract and milk) even at temperatures below the minimal growth temperature. All B. cereus strains tested yielded positive results with a commercial test kit for diarrhoeal enterotoxin. The results indicate that strains with the capacity to grow at temperatures < or = 7 degrees C are not essentially different from those with minimal growth temperatures of > 10 degrees C.

Effect of Sodium Lactate on Toxin Production, Spore Germination and Heat Resistance of Proteolytic Clostridium botulinum Strains.

The effect of sodium lactate and sodium lactate combined with sodium chloride (NaCl) on toxin production by proteolytic strains of Clostridium botulinum was determined in peptone-yeast extract medium, pH 6.1. Both inhibitors were also tested for their effect on thermal destruction of spores. Additionally, the effect of sodium lactate on germination of spores was assessed. The inhibitory effect of sodium lactate was dependent on the applied incubation temperature. The best inhibition was obtained at low temperatures. Toxin production was delayed at 15 and 20°C by sodium lactate concentrations of 2 and 2.5%, respectively. Complete inhibition of toxin production at 15, 20 and 30°C occurred at concentrations of 3, 4 and >4%, respectively. Further, sodium lactate inhibited germination of the C. botulinum spores, which may partially explain the inhibitory effect of sodium lactate on growth and toxin formation. The inhibitory effect of NaCl at concentrations resulting in an identical water activity value as obtained by sodium lactate was negligible, indicating that the inhibitory effect of sodium lactate was not caused by decreasing water activity. No clear synergistic effect of sodium lactate (1.5 or 2.5%) and NaCl (2.1%) was observed.

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.

Feasibility of a reference material for staphylococcal enterotoxin A.

A reference material for staphylococcal enterotoxin A (SEA), was produced by spray-drying the toxin in milk. With this procedure the SEA was distributed homogeneously in the material. For ease of handling the reference material was encased in gelatin capsules, each containing 405 ng of SEA. Simply dissolving the milk powder in distilled water resulted in a 100% recovery of the SEA present. The reference material would appear suitable for testing laboratory performance, comparison of detection methods and to validation of extraction procedures.

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.

DNA hybridization and latex agglutination for detection of heat-labile- and shiga-like toxin-producing Escherichia coli in meat.

DNA-hybridization and latex-agglutination tests were used for screening of a group of Escherichia coli isolates for heat-labile enterotoxin (LT)- and shiga-like toxin (SLT1 or VT1) -producing strains, respectively. Strains tested originated from 162 meat samples (poultry, pigs and beef) chosen at random. Additionally LT- and SLT1-producing reference strains were tested. The DNA-hybridization technique allowed screening of large numbers of strains, whereas large scale testing of strains by latex agglutination was laborious. Of 800 E. coli strains tested by DNA hybridization none contained the gene encoding LT. Production of LT as tested by latex agglutination was not found. The gene encoding SLT1 was detected in 10 of the 800 isolates tested. None of these strains, however, showed cytotoxicity on Vero cells. Serotyping was done with sorbitol-negative E. coli strains, first by using the latex-agglutination test for O157 followed by complete serotyping. No E. coli of serogroup O157 were found. Therefore the results obtained also indicate that routine screening of E. coli isolated randomly from food for toxin production is not useful and should be limited to food-borne disease outbreaks with an etiology resembling an E. coli infection.

Botulism Risk of Refrigerated, Processed Foods of Extended Durability.

Control of the botulism risk in refrigerated, processed foods with extended durability (REPFED) which do not contain intrinsic safety factor(s) has been analyzed. There are insufficient data on the heat resistance of spores of nonproteolytic Clostridium botulinum types B, E, and F, to ensure that the heating process used in preparation of REPFEDs provides adequate lethality. During portioning of foods and filling of trays, products may be exposed to recontamination if this process is not carried out under aseptic conditions. Pasteurization of sealed trays at a temperature of about 75°C for several minutes is a process which spores of C. botulinum can easily survive. For these reasons, REPFEDs must be stored at a temperature < 3.3°C, since such low storage temperatures prevent growth and formation of toxin by C. botulinum . At 8°C, a temperature to which chilled foods are often exposed during and after retail sale, nonproteolytic strains of C. botulinum can produce toxin within 3 weeks. In addition prestorage at 3°C for up to 2-4 weeks stimulates the toxinogenesis of nonproteolytic C. botulinum type B at a subsequent storage at 8°C. Heating of REPFEDs before consumption was not always sufficient to inactivate botulinum toxin completely. In order to ensure that the risk of botulism from these foods is controlled adequately, REPFEDs must be stored at a temperature < 3.3°C. If, however, this temperature cannot be guaranteed, the storage time has to be limited.

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.