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 at 7°C and consumed within the "best before" date, this will not cause any problems for healthy adults.

In order to determine the major contamination sources of raw milk with (psychrotrophic) Bacillus cereus, the incidence of B. cereus spores and vegetative cells on farms in the Netherlands was investigated. Samples were taken from air, soil, grass, bedding (used and unused), feed (hay, silage, concentrate, beet), drinking water, faeces, udders and milk. Some biochemical and growth characteristics of the strains isolated were examined. In total 847 presumptive B. cereus colonies were isolated from all sources examined. The levels present in the various samples ranged from < 10 up to 10(7) per g or ml for vegetative cells and < 10 to 10(5) per g or ml for spores. According to the ISO confirmation tests and/or the carbohydrate patterns (API 50 CHB), 766 (90%) of these isolates were confirmed to be B. cereus. The carbohydrate patterns revealed more than 30 different B. cereus types. No distinct relation between the biochemical characteristics and the contamination source was observed. The major contamination sources of B. cereus were soil and faeces. In winter when cows are housed, used bedding probably also participates in this contamination route. The udder will be contaminated, finally resulting in the presence of these organisms in raw milk. About 40% of the isolated strains showed growth at 7 degrees C and were considered to be psychrotrophic. Of the strains isolated from raw milk 30% were able to grow at 7 degrees C. The presence of psychrotrophic spores in raw milk will lead to their presence in pasteurized milk due to heat-resistance properties. Since at low storage temperatures these psychrotrophs will germinate, grow and subsequently spoil the product faster than mesophilic types, the psychrotrophic strains will have a more significant impact on the keeping quality of milk.

Listeria monocytogenes is a gram-positive, psychotrophic, food-borne pathogen which is able to grow in osmotically stressful environments. Carnitine (beta-hydroxy-L-tau-N-trimethyl aminobutyrate) can contribute significantly to growth of L. monocytogenes at high osmolarity (R. R. Beumer, M. C. te Giffel, L. J. Cox, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 60:1359-1363, 1994). Transport of L-[N-methyl-14C]carnitine in L. monocytogenes was shown to be energy dependent. Analysis of cell extracts revealed that L-carnitine was not further metabolized, which supplies evidence for its role as an osmoprotectant in L. monocytogenes. Uptake of L-carnitine proceeds in the absence of a proton motive force and is strongly inhibited in the presence of the phosphate analogs vanadate and arsenate. The L-carnitine permease is therefore most likely driven by ATP. Kinetic analysis of L-carnitine transport in glucose-energized cells revealed the presence of a high-affinity uptake system with a Km of 10 microM and a maximum rate of transport (Vmax) of 48 nmol min-1 mg of protein-1. L-[14C]carnitine transport in L. monocytogenes is significantly inhibited by a 10-fold excess of unlabelled L-carnitine, acetylcarnitine, and tau-butyrobetaine, whereas L-proline and betaine display, even at a 100-fold excess, only a weak inhibitory effect. In conclusion, an ATP-dependent L-carnitine transport system in L. monocytogenes is described, and its possible roles in cold adaptation and intracellular growth in mammalian cells are discussed.

The effect of environmental conditions on the germination of Bacillus cereus was investigated Germination was affected to varying extent by strain type, complexity of the medium, presence of germinants and temperature. in the microbiological examination of food (ingredients) if was shown that germination during sample preparation is observed for all bacterial spores present in these products. in standard procedures for microbiological examination it is mentioned that the time between preparation of the initial suspension and the mixing of dilutions and media shall not exceed 45 min. However, in this study if was shown that in 45 min significant numbers of spores have already germinated. Therefore, too much time between preparation of the primary dilution and the heat activation step led to an underestimation of the numbers of spores. in order to determine the proportion of spore-forming bacteria present as spores, the time between preparation of the primary dilution and the heat activation step should be as short as possible (<10 min) and the temperature of diluents during the analysis should be as low as possible (e.g in melting ice). (C) 1995 Academic Press Limited

Faeces and raw milk from individual cows were examined for the presence of Campylobacter jejuni. After drawing milk, the lactoperoxidase system was inactivated by raising the pH to 7.5. The organism was isolated from 22% of 904 faecal samples and from 4.5% of 904 milk samples. From laboratory experiments it could be concluded that inactivation of the lactoperoxidase system resulted in a better isolation of C. jejuni from raw cows' milk.

Uit steekproeven blijkt de microbiologische kwaliteit van filet americain, waarin rauw varkensvlees is verwerkt, zodanig dat consumptie ontraden kan worden. Dit geldt zowel voor als het produkt door de detaillist is bereid, als wanneer het product industrieel is bereid

Summary One hundred and eighty-two raw, 112 pre-cooked and 750 cooked hamburgers composed mainly of beef or beef and pork were subjected to microbiological examination. Raw hamburgers gave total bacterial counts from 105 to 108 per g, counts of Enterobacteriaceae from 104 to 108 per g, of Escherichia coli from 103 to 105, of group D streptococci from 102 to 104, of Staphylococcus aureus from 3 to 102 and of Clostridium perfringens less than 10 bacteria per g. Of the samples, 32 % contained salmonellas; the highest most probable number was 102 per g but most estimates were below 1 per g. Corresponding figures for the pre-cooked samples were 2–3 log cycles lower, and only one sample contained salmonella. Yersinia enterocolitica was not isolated from any raw or pre-cooked sample. Three hundred and ninety-five of the cooked hamburgers were prepared by grilling raw hamburgers for between 2 and 5·5 min. These gave total bacterial counts from 105 to 107 per g, and counts of Enterobacteriaceae from 102 to 105 per g. Of the samples, 9·4 % contained salmonellas, always in numbers below 1 per g. The remaining 355 cooked hamburgers were prepared from samples pre-cooked for 10 min at 80 °C. Some were grilled and some fat fried. The total bacterial counts were from 103 to 105 per g, and counts of Enterobacteriaceae below 102 perg. Salmonellae, again in small numbers only, were recovered from 3·5 % of samples. When hamburgers were artificially contaminated with Salmonella typhimurium it took 5·5 min on a commercial grill, 2·25 min frying in a frying pan and 1·75 min on a household grill to reliably reduce the salmonella count one hundredfold. This means that at many vending places hamburgers are often cooked for too short a time. D-values were determined for S. typhimurium in hamburger meat at 50, 55, 60, 65 and 70 °C, these values were 7·1, 5·1, 1·2, 0·9 and 0·6 min respectively. It can be concluded that the heating action in the centre of the hamburgers will take place more slowly than in the hamburger as a whole, and that the time between cooking and consumption is very important in reducing the microbial load to acceptable levels. Pre-cooking (10 min at 80 °C in a water bath) gives a reduction in the numbers of salmonella of about 4 × 103, after which cooking gives a further reduction as mentioned above.

Experiments were carried out to assess the reduction rate of two salmonella strains (S. eastbourne and S. typhimurium) in chocolate bars. After artificial contamination of chocolate, after 'conching', with about 10(6) S. eastbourne/g. this organism was still recovered after 9 months storage. The strain of S. typhimurium was less resistant. Both serotypes died off more rapidly in bitter chocolate than in milk chocolate. After contamination with a smaller dose (about 10(3)/g.) with these two serotypes, similar differences were observed.