The Bacillus cereus Group comprises organisms that are widely distributed in the environment and are of health and economic interest. We demonstrate an 'ecotypic' structure of populations in the B. cereus Group using (i) molecular data from Fluorescent Amplified Fragment Length Polymorphism patterns, ribosomal gene sequences, partial panC gene sequences, 'psychrotolerant' DNA sequence signatures and (ii) phenotypic and descriptive data from range of growth temperature, psychrotolerance and thermal niches. Seven major phylogenetic groups (I to VII) were thus identified, with ecological differences that provide evidence for a multiemergence of psychrotolerance in the B. cereus Group. A moderate thermotolerant group (VII) was basal to the mesophilic group I, from which in turn distinct thermal lineages have emerged, comprising two mesophilic groups (III, IV), an intermediate group (V) and two psychrotolerant groups (VI, II). This stepwise evolutionary transition toward psychrotolerance was particularly well illustrated by the relative abundance of the 'psychrotolerant' rrs signature (as defined by Pruss et al.) copies accumulated in strains that varied according to the phylogenetic group. The 'psychrotolerant' cspA signature (as defined by Francis et al.) was specific to group VI and provided a useful way to differentiate it from the psychrotolerant group II. This study illustrates how adaptation to novel environments by the modification of temperature tolerance limits has shaped historical patterns of global ecological diversification in the B. cereus Group. The implications for the taxonomy of this Group and for the human health risk are discussed.

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.Abbreviations: FTIR, Fourier transform Infrared; Hbl, haemolysin BL; MLST, multilocus sequence typing; Nhe, non-haemolytic enterotoxin; RAPD, random amplification of polymorphic DNA.The GenBank/EMBL/DDBJ accession numbers for the sequences of the internal gene fragments used for MLST and for the sporulation stage III AB genes reported in this paper are AY762151-AY762213 and AY578317-AY578349, respectively.

Psychrotrophic Bacillus cereus is a limiting factor for the shelf-life of pasteurized milk, particularly during the grazing season. Potential sources of contamination and factors that might affect the spore content of milk were studied in detail for a group of eight cows during three 2-wk study periods from June to September over 2 yr. The spore content of milk was strongly associated with the degree of contamination of the teats with soil. High water content of soil, low evaporation of water and dirty access alloys were the most important factors correlating with high spore concentrations. The spore content of soil varied from < 50 to 380,000/g, depending on time and sampling site. The milking equipment did not contribute significantly to the contamination. The spore contents in air during milking (< 100 cfu/m3) and in feed (silage, hay, fresh grass, and concentrates) were too low to be of importance for contamination. The spore content in dung was also low. Further support that soil was the major contamination source was found by comparison of genetic fingerprints by random amplified polymorphic DNA polymerase chain reaction of isolates of B. cereus from soil and milk and by teat cleansing experiments, which resulted in reduced contamination levels in milk.

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.

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An important question in conservation biology is whether the biodiversity of different taxa is correlated. We studied the extent to which the number of species of six different taxa—plants, birds, butterflies, bumblebees, ground beetles, and dung beetles—in 31 Swedish seminatural grasslands covary, and whether species diversity can be related to habitat variables. During 1996 and 1997, we surveyed plants and animals with appropriate techniques for each taxa and mapped the grassland habitat. In general, correlations between taxa were few. Grassland plant diversity (currently used as an indicator for conservation value) was only significantly positively correlated to total bird diversity. Bumblebee diversity was significantly positively related to both total and grassland butterflies, whereas there was a significant negative relationship between grassland birds and dung beetles. Plants, birds, bumblebees, and butterflies showed significant similarities in patterns of species composition, as did birds, butterflies, grassland butterflies, and ground beetles. The total number of plants and both subsets of birds (total and grassland) were significantly positively related to area, whereas there was a significant negative association between area and dung‐beetle diversity. The diversity of both butterflies and bumblebees was significantly negatively related to the proportion of short‐grazed field layer. Bumblebees showed a positive relationship with junipers, whereas ground beetles and grassland birds were negatively associated with trees. The total number of bird species was positively influenced by the occurrence of shrubs. Our results suggest that neither the species richness of grassland plants nor that of any other of the surveyed taxa can be used as an indicator for total biodiversity in seminatural grasslands. The lack of similar patterns of species composition among taxa also makes it difficult to define functional groups with similar habitat demands. Until we have more detailed knowledge of the demands of species and taxa, it is important that we direct management efforts so that we provide a wide spectrum of grassland characteristics.

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n = 17), strains connected to diarrheal foodborne outbreaks (n = 40) and food-environment strains (n = 43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 °C. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 °C and 49% at 7 °C (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 °C, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxinproducing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 °C and a lower germination, particularly at 7 °C, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 °C, 37 °C, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.

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