Isolation, identification and characterization of Bacillus cereus from the dairy environment

In this thesis the occurrence of Bacillus cereus in the milk production and processing environment was investigated. Isolates were identified biochemically and by DNA probes based on the variable regions of 16S rRNA. Further characterization was carried out using biochemical and molecular typing, in order to determine the major contamination sources of milk. Furthermore, properties in relation to carbohydrate utilization, growth at low temperatures and enterotoxin production were examined.B. cereus is important as food spoilage organism. In the present study the microorganism was isolated from food ingredients such as yeast, flour, cacoa, herbs and spices. B. cereus was also found in a wide variety of processed food products including bakery products, Chinese meals, pasta products, chocolate and meat products. In pasteurized milk and dairy products, B. cereus was frequently present and it is well-known that it can be responsible for spoilage when post-heat- treatment contamination is absent.To enumerate spores in a sample, the most common procedure is to carry out a heat- activation treatment of 10 min at 80°C, followed by plating on a (selective) agar medium. To prevent germination of spores during sample preparation the time between the preparation of the primary dilution and heat-activation step should be less than 10 min and the temperature during the analysis should be as low as possible (e.g. by keeping dilutions in melting ice). After isolation, presumptive B. cereus are confirmed by biochemical tests, however, this may lead to incorrect identification. Several isolates, involved in food poisoning incidents, were shown to be B. thuringiensis, by sequencing part of the 16S rRNA. These results suggest that use of B. thuringiensis as insecticide may lead to foodborne infection or intoxication. To improve the confirmation procedure, we developed a specific and sensitive method, using DNA probes based on variable regions of the 16S rRNA, to differentiate between B. cereus and B. thuringiensis.On farms, B. cereus is introduced into raw milk by contamination of the udder with faeces, soil and, in winter, used bedding. In the dairy processing plants, additional contamination takes place via the equipment. Biochemical and growth characterization and molecular typing of isolates confirmed this and also showed that selection of strains occurs in the milk production and processing chain. Cleaning and disinfection will not eliminate all B. cereus in milking installations or heat exchangers, particularly not those adhering to surfaces of the equipment.Although only a few cases of milkborne infection and intoxication by B. cereus have been reported, most isolates were able to produce enterotoxin as determined by immunoblotting, cytotoxicity tests and PCR. However, if pasteurized milk is stored at7°C and consumed within the "best before" date, this will not cause any problems for healthy adults.