Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp

AIMS: To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 and nisin were added to brain heart infusion (BHI) medium (40-160 AU ml(-1)) or mango pulp (100 AU ml(-1)) and the growth of Bacillus cereus and Bacillus thuringiensis was monitored. Cultures treated with bovicin HC5 or nisin showed longer lag phases and grew slower in BHI medium. Bovicin HC5 and nisin were bactericidal and showed higher activity in mango pulp at acidic pH values. To determine the effect on spore germination and D values, mango pulp containing bovicin HC5 was inoculated with 10(6) and 10(9) spores per ml(-1), respectively, from each strain tested. Bovicin HC5 reduced the outgrowth of spores from B. cereus and B. thuringiensis, but thermal sensitivity was not affected. CONCLUSIONS: Bovicin HC5 was bactericidal against B. cereus and B. thuringiensis isolated from spoiled mango pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus and B. thuringiensis had not been previously isolated from spoiled mango pulp and bovicin HC5 has the potential to inhibit such bacteria in fruit pulps.     

RAPID DETECTION OF GERMINATING BACILLUS CEREUS CELLS USING FLUORESCENT IN SITU HYBRIDIZATION

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.
B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus -specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 10³ cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.     

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.     

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.     

Characterization of emetic Bacillus weihenstephanensis, a new cereulide-producing bacterium

Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8 degrees C.     

Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.     

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0. CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.     

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil

AIMS: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. METHODS AND RESULTS: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. CONCLUSION: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.     

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures.

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.     

Development of a bioactive packaging cellophane using Nisaplin as biopreservative agent

AIMS: Production of a nisin-containing cellophane-based coating to be used in the packaging of chopped meat. METHODS AND RESULTS: The adsorption of nisin to cellophane 'P' type surface was studied at 8, 25, 40 and 60 degrees C using different concentrations of nisin. Then, the antimicrobial activity of adsorbed nisin to cellophane surface was determined in fresh veal meat for effectiveness in reducing the total aerobic bacteria. The adsorption of nisin to cellophane was higher at 8 degrees C. The developed bioactive cellophane reduced significantly the growth of the total aerobic bacteria (by ca 1.5 log units) through 12 days of storage at 4 degrees C. CONCLUSIONS: Bioactive cellophane packaging could be used for controlling the microbial growth in chopped meat. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-adsorbed bioactive cellophane would result in an extension of the shelf life of chopped meat under refrigeration temperatures.     

A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification

AIMS: The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non-Bradyrhizobium Endophytic Bacterium 17. METHODS AND RESULTS: The bacterial peptide was analysed and purified via HPLC. Using the disk diffusion assay this peptide inhibited the growth of 16/19 B. thuringiensis strains, 4/4 Bacillus cereus strains, among others, as well as a Gram-negative strain Escherichia coli MM294 (pBS42). Both bactericidal and bacteristatic effects were observed on B. cereus ATCC 14579 and bactericidal effects were observed on B. thuringiensis ssp. thuringiensis Bt1267. The molecular weight of the peptide was estimated via SDS-PAGE and confirmed with Matrix Assisted Laser Desorption Ionization Quadrapole Time of Flight mass spectrometry; its weight is 3162 Da. The peptide is biologically active after exposure to 100 degrees C for 15 min, and within the pH range 1.00-9.25. Its activity disappeared when treated with proteinase K and protease, but not with alpha-amylase or catalase. CONCLUSIONS: We conclude that this is the first report of a bacteriocin produced by a plant growth promoting rhizobacteria (B. thuringiensis) species and have named the bacteriocin thuricin 17. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work has characterized a bacteriocin produced by a plant growth promoting bacterium. This strain is previously reported to increase soya bean nodulation.     

Characterization of spore appendages from Bacillus cereus strains

AIMS: Further characterization and comparison of spore appendages from Bacillus cereus strains. METHODS AND RESULTS: Appendages were isolated from 10 B. cereus strains from the food industry and food-borne outbreaks. The appendage proteins were dissolved in sample buffer containing 2% SDS and 5% mercaptoethanol at 100 degrees C, and subjected to SDS-PAGE. None of the appendages showed identical protein patterns. Western blots, using antibodies raised against a 3.5 kDa appendage protein, showed that the majority of the appendage proteins reacted with the antibody. Removal of the appendages by sonic treatment of the spores did not alter their heat resistance. The appendages were digested by proteinase K, pepsin, and the enzymes in the detergent Paradigm 10, but not by trypsin or chymotrypsin. Spore adhesion to stainless steel was scarcely affected by removal of the appendages. Digestion of adhered intact spores (with appendages) with Paradigm 10 showed a high degree of variation. CONCLUSIONS: Spore appendages from B. cereus are complex proteinaceous structures that differ among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Information about spore appendages and their involvement in spore adhesion is crucial for improving cleaning methods used for control of bacterial spores in the food industry.     

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

AIMS: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). METHODS AND RESULTS: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. CONCLUSIONS: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. SIGNIFICANCE AND IMPACT OF THE STUDY: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.     

Salt extends the upper temperature limit for growth of food-poisoning bacteria

Inclusion of NaCl into the growth medium raised the upper temperature limit of growth of the following organisms: Staphylococcus aureus (two strains), Salmonella senftenberg, S. typhimurium, Escherichia coli, Streptococcus faecalis, Bacillus cereus, Clostridium sporogenes, C. perfringens (two strains). The magnitude of the response varied with the culture, the largest being 3.5 degrees with B. cereus cells. The spores of B. cereus were not protected by salt but clostridial spores behaved as the vegetative cells (response of 2.5 degrees). The optimal salt concentration for the protective effect varied with the organism ranging from 0.2 M for the Gram-negative organisms to 1.0 M for S. aureus.