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.

Minimally processed fresh (MPF) fruits and vegetables are good media for growth of microorganisms. They have been involved in outbreaks because of the consumption of products contaminated by pathogens. They are also sensitive to various spoilage microorganisms such as pectinolytic bacteria, saprophytic Gram-negative bacteria, lactic acid bacteria, and yeasts. Contamination of MPF fruits and vegetables occurs at every stage of the food chain, from cultivation to processing. Polluted environments during cultivation or poor hygienic conditions in processing increase the risk of contamination with foodborne pathogens. Although MPF fruits and vegetables may harbor psychrotrophic microorganisms such as fluorescent pseudomonads or Listeria monocytogenes, good control of refrigeration temperature limits growth of spoilage and pathogenic microorganisms. Modified atmospheres are often efficient to maintain or improve visual organoleptic quality of MPF fruits and vegetables, but their effects on microorganisms are inconsistent. Chemical disinfection can partially reduce the initial bacterial contamination; irradiation seems to be more efficient. The applications of legislations and quality assurance systems to control contamination, survival, and growth of foodborne pathogens in MPF fruits and vegetables are discussed.

The enterotoxigenic profiles of 51 B. cereus food-related strains were compared to those of 37 B. cereus food-poisoning strains. cytK and association of hbl-nhe-cytK enterotoxin genes were more frequent among diarrheal strains (73 and 63%) than among food-borne strains (37 and 33%). Unlike diarrheal strains, food-borne strains showed frequent nhe and hbl gene polymorphisms and were often low toxin producers.

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.

An aerobic endospore-forming bacillus ) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97 % similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95 %. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C 15 : 0 , C 16 : 0 , iso-C 17 : 0 , anteiso-C 15 : 0 , iso-C 16 : 0 , iso-C 13 : 0 ) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98 T taxon was more specifically characterized by an abundance of iso-C 15 : 0 and low amounts of iso-C 13 : 0 compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98 T taxon from the six current B. cereus Group species.NVH 391-98 T therefore represents a novel species, for which the name Bacillus cytotoxicus sp. Abbreviations: DDH, DNA-DNA hybridization; MLST, multilocus sequence typing.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene of strains are described in Tables 1 and S1. The sequences of the MLST genes can be found at http://mlstoslo.uio.no/index.html.A supplementary figure and three supplementary tables are available with the online version of this paper.

Cytotoxic activity levels of culture filtrates and toxin distributions varied according to the phylogenetic group (I to VII) within the Bacillus cereus group, suggesting that these groups are of different clinical significance and are more suitable than species affiliations for determining food poisoning risk. A first-line, simple online tool (https://www.tools.symprevius.org/Bcereus/english.php) to assign strains to the different phylogenetic groups is presented.

A rosemary extract commercially exploited (Oxy'less) as an antioxidant of lipids in foods was dissolved in ethanol (100 mg/ml), and the solution was tested against foodborne microorganisms. For gram-positive bacteria, the MIC of the ethanolic solution was 1% for Leuconostoc mesenteroides, 0.5% for Listeria monocytogenes, 0.5% for Staphylococcus aureus, 0.13% for Streptococcus mutans, and 0.06% for Bacillus cereus. It slowed the growth of Penicillium roquefortii and Botrytis cinerea. Up to 1% of the ethanolic solution had no activity on the gram-negative bacteria Escherichia coli, Salmonella Enteritidis, and Erwinia carotovora and on the yeasts Rhodotorula glutinis and Cryptococcus laurentii. Antibacterial activity of the rosemary extract was strongly influenced by the composition of the media. The MIC was reduced by low pH, high NaCl contents, and low temperatures. Low pH and high NaCl concentration had a synergistic effect on the MIC of the rosemary extract for S. aureus. Lipids, surface-active agents, and some proteins decreased its antibacterial activity, whereas pectin had no effect. The inhibitory effect was little modified by heat treatment (100 degrees C). The natural microflora of pasteurized zucchini broth was inhibited by 0.5% of the rosemary extract. The antibacterial activity was linked to the compounds extracted with hexane, which are presumably phenolic diterpenoids.

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.