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

Modelling Contamination of Raw Milk with Butyric Acid Bacteria Spores

Raw milk contains low concentrations of bacterial endospores, originating from the farm environment (e.g. soil, feeds, faeces). Spores of Clostridium tyrobutyricum, also called butyric acid bacterium (BAB), are of great interest to the dairy industry. They survive milk pasteurisation and cause off-flavours and texture defects in various cheese types. The contamination pathway of BAB spores is well known. Their primary origin is soil. In silage the number of spores will increase if conditions permit BAB growth. The spores are excreted in the cows faeces and are transferred to milk by contaminated teat surfaces. Many factors are involved in the contamination of milk with BAB spores. In this study, the contamination pathway was described using a combination of predictive models. The objective of the study was to quantitatively assess the importance of the different steps of the contamination pathway and to identify the most effective control points

Predictive Modelling Of Temperature And Water Activity (Solutes) On The In Vitro Radial Growth Of Botrytis Cinerea Pers

peer reviewe

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed

Effective Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Raw Milk Contaminated with Naturally Infected Feces▿

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 102 to 3.5 × 105 cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90°C at holding (mean residence) times of 6 to 15 s. Following 72°C and a holding time of 6 s, 70°C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an Ea of 305,635 J/mol and an lnk0 of 107.2, corresponding to a D value of 1.2 s at 72°C and a Z value of 7.7°C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at ≥72°C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis