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Bacteria from the Bacillus cereus group exhibit genetic and physiological diversity through different ecotypes. Here, we present the draft genome sequences of 20 bacterial strains belonging to the contrasted psychrotolerant and thermotolerant ecotypes.

Isolates of the Bacillus cereus group (396 in total) from farms, silo tanks and production lines for pasteurised milk were tested for toxin production potential, and by polymerase chain reaction (PCR) for the presence of toxin genes. Comparison between the tests indicated the presence of gene polymorphisms. Highly toxigenic strains, based on production of subunit A of the nonhemolytic enterotoxin, NHE (NheA) and subunit C of the haemolytic enterotoxin, HBL (HblC), were less common among dairy isolates compared with farm and silo isolates. No producer of high levels of both toxins was found among 156 psychrotrophic dairy isolates (B. weihenstephanensis) and only 3% of all psychrotrophs were high producers of NheA. Psychrotrophic B. cereus from pasteurised milk appeared to have a low enterotoxin production potential, and they were not producers of emetic toxin or cytotoxin K and therefore may be less likely to cause illness than mesophilic strains.

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.

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.

International audience; Hydrosols are hydrodistillation products of aromatic plants. They contain less than 1 g/L of dispersed essential oils giving organoleptic properties. Hydrosols are subjected to microbial proliferation. Reasons for spoilage have to be found in the nature of substrates supporting growth and ofmicrobiological contaminants. The composition in essential oils and themicrobiota of 22 hydrosol samples of Citrus aurantium L. ssp. amara L. (orange blossom), Rosa damascena Miller (rose D.), and Rosa centifolia L. (rose C.) flowers were analyzed to determine the factors responsible for decay. The median concentrations in essential oils were 677 mg/L for orange blossom hydrosols, 205 mg/L for rose D. hydrosols, and 116 mg/L for rose C. hydrosols. The dry matter content of these hydrosols varied between 4.0 mg/L and 702 mg/L, and the carbohydrate content varied between 0.21 mg/L and 0.38 mg/L. These non-volatile compounds were likely carried over during distillation by a priming and foaming effect, and could be used as nutrients by microorganisms. A microbial proliferation at ambient temperature and also at 5 °C has been observed in all studied hydrosols when stored in a non-sterile container. In contaminated hydrosols,maximal countswere about 7 log10 CFU/mL,while the French pharmacopeia recommends a maximal total bacterial count of 2 log10 CFU/mL. Neither yeast nor mold was detected. The isolated microbial population was composed of environmental Gram-negative bacteria, arranged in four major genera: Pseudomonas sp., Burkholderia cepacia complex, and presumably two new genera belonging to Acetobacteraceae and Rhodospirillaceae. Among those bacteria, Burkholderia vietnamiensis and Novosphingobium capsulatum were able to metabolize volatile compounds, such as geraniol to produce 6-methyl-5-hepten-2-one or geranic acid, or phenylethyl acetate to produce 2-phenylethanol. EO concentrations in hydrosols or cold storage are not sufficient to insure microbiological stability. Additional hurdles such as chemical preservatives or aseptic packaging will be necessary to insure microbial stability.

International audience; Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery.

International audience; Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Background The Bacillus cereus Group consists of closely-related bacteria, including pathogenic or harmless strains, and whose species can be positioned along the seven phylogenetic groups of Guinebretière et al. (I–VII). They exhibit different growth-temperature ranges, through thermotolerant to psychrotolerant thermotypes. Among these, B. cytotoxicus is an atypical thermotolerant and food-poisoning agent affiliated to group VII whose thermotolerance contrasts with the mesophilic and psychrotolerant thermotypes associated to the remaining groups I–VI. To understand the role of fatty acid (FA) composition in these variable thermotypes (i.e. growth behavior vs temperatures), we report specific features differentiating the FA pattern of B. cytotoxicus (group VII) from its counterparts (groups I–VI). Findings The FA pattern of thermotolerant group VII (B. cytotoxicus) displayed several specific features. Most notably, we identified a high ratio of the branched-chain FAs iso-C15/iso-C13 (i15/i13) and the absence of the unsaturated FA (UFA) C16:1(5) consistent with the absence of ∆5 desaturase DesA. Conversely, phylogenetic groups II–VI were characterized by lower i15/i13 ratios and variable proportions of C16:1(5) depending on thermotype, and presence of the DesA desaturase. In mesophilic group I, thermotype seemed to be related to an atypically high amount of C16:1(10) that may involve ∆10 desaturase DesB. Conclusion The levels of i15/i13 ratio, C16:1(5) and C16:1(10) UFAs were related to growth temperature variations recorded between thermotypes and/or phylogenetic groups. These FA are likely to play a role in membrane fluidity and may account for the differences in temperature tolerance observed in B. cereus Group strains. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1288-4) contains supplementary material, which is available to authorized users.

Pas de clé UT; The large bacterial genus Bacillus is widely distributed in the environment and is able to colonize highly diverse niches. Some Bacillus species harbor pathogenic characteristics. The fatty acid (FA) composition is among the essential criteria used to define Bacillus species. Some elements of the FA pattern composition are common to Bacillus species, whereas others are specific and can be categorized in relation to the ecological niches of the species. Bacillus species are able to modify their FA patterns to adapt to a wide range of environmental changes, including changes in the growth medium, temperature, food processing conditions, and pH. Like many other Gram-positive bacteria, Bacillus strains display a well-defined FA synthesis II system that is equilibrated with a FA degradation pathway and regulated to efficiently respond to the needs of the cell. Like endogenous FAs, exogenous FAs may positively or negatively affect the survival of Bacillus vegetative cells and the spore germination ability in a given environment. Some of these exogenous FAs may provide a powerful strategy for preserving food against contamination by the Bacillus pathogenic strains responsible for foodborne illness.

Phylogeny of the B. cereus Group based on rrs and ITS1 gene sequence analysis, and position of species in this phylogeny. This file illustrates the position of the B. cereus sl species over 7 major phylogenetic groups according to the classification scheme given in Guinebretière et al. 2008. It also shows the three main branches of evolution in the B. cereus Group.

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.

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature ( T min ). CasKR was also involved in optimal cold growth above T min and in cell survival below T min . Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a Δ casKR mutant restored its ability to grow at T min . Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.

International audience; The growth rates of strains covering the seven major phylogenetic groups of Bacillus cereus sensu lato (as defined by Guinebretiere et al., 2008) at a range of temperature (7 Ce55 C), pH (4.6e7.5) and a w (0.929e0.996, with 0.5%e10% NaCl as humectant) were determined. Growth rates were fitted by non-linear regression to determine the cardinal parameters T min , T opt , T max , pH min , pH opt , a wmin and m opt. We showed that cardinal parameters reflected the differences in the temperature adaptation observed between B. cereus phylogenetic groups I to VII. The ability of growing at low pH (up to 4.3) or low a w (from a w 0.929 and up to 10% NaCl) varied among strains. The strains of groups III and VII, the most tolerant to heat, were also the most adapted to high NaCl (all strains growing at 8% NaCl) and the ones of groups I and VI the least adapted (no growth at 7% NaCl). All strains of groups II and VII were able to grow at pH 4.6, and only a few strains of group VI. Phenotypic differences between the two psy-chrotrophic groups II and VI were revealed by contrasted acid and salt tolerance. The cardinal values determined in this work were validated by comparing with cardinal parameters of a panel of strains published elsewhere and with predictions of growth in a range of foods.

An aerobic endospore-forming bacillus (NVH 391-98T) 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-C15 : 0, C16 : 0, iso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C13 : 0) supported the affiliation of these strains to the genus Bacillus , and more specifically to the B. cereus Group. NVH 391-98T taxon was more specifically characterized by an abundance of iso-C15 : 0 and low amounts of iso-C13 : 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 391-98T taxon from the six current B. cereus Group species. NVH 391-98T therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98T ( = DSM 22905T = CIP 110041T).

Cytotoxic activity levels of culture filtrates and toxin distributions varied according to the phylogenetic group (I to VII) within the B acillus 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.

Bacillus cereus is found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing to B. cereus pathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57 B. cereus strains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulence in vivo assays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that the hlyII gene is carried only by strains with a pathogenic potential and that the expression levels of inhA1 and nprA are higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of various B. cereus strains.

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalloproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1 , inhA2 , and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system.

Consequences of climate change on the ecology of pathogens are difficult to forecast. However changes affecting microorganisms will likely involve already known evolution or adaptation mechanisms. Bacillus cereus is a frequent cause of foodborne poisonings and is known as a soil borne bacterium. B. cereus may represent an interesting model to study the impact of climate change on foodborne pathogens. The B. cereus group (or B. cereus sensu lato) displays a wide diversity of strains recently distributed in seven major phylogenetic groups. B. cereus growth domains range from psychrotrophic to nearly thermophilic. Current climate selects B. cereus distribution: psychrotrophes are more common in cold areas, while mesophiles prevail in tropical soils. In response to external signals, B. cereus may adapt to changing environments by varied mechanisms. Some illustrations of the signal transduction systems (two-component systems, alternative r factors) and of the mechanisms of B. cereus adaptation to major environmental factors (temperature, carbon source, redox potential and pH) are proposed. The environment of sporulation has an impact on spore properties; heat resistance is positively correlated with sporulation temperature. Surveillance needed to detect changes in the epidemiology of B. cereus foodborne poisonings as a consequence of climate change is discussed.

The Bacillus cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about 30 clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to Bacillus anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the B. cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain should be a representative of a novel bacterial species, for which the name Bacillus cytotoxis or Bacillus cytotoxicus is proposed. This strain has a reduced genome size compared to other B. cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical facultative psychrophile generally found in soil. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrophile strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome, or core genome, in the B. cereus group to be 3000+/-200 genes and the total proteome, or pan-genome, to be 20-25,000 genes.

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.

Background Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. Results A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. Conclusion Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel nhe operon and the cytK-1 gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other B. cereus group strains.

International audience; Haemolysin BL (HBL) and nonhaemolytic 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–L2 and NheB component ranged from 0.02 to 5.6 mgmL 1 and from 0.03 to 14.2 mgmL 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.

A food processing plant producing pasteurized purees and its zucchini puree processing line were examined for contamination with aerobic and facultative anaerobic bacterial spores during a day's operation. Multiplication of spores was also monitored in the product stored under different conditions. High concentrations of Bacillus cereus spores were found in the soil in which the zucchinis were grown (4.6+/-0.3 log CFU/g), with a background spore population of 6.1+/-0.2 log CFU/g. In the processing plant, no B. cereus or psychrotrophic bacterial spores were detected on equipment. B. cereus and psychrotrophic bacterial spores were detected after enrichment in all samples of raw zucchinis, washed zucchinis, of two ingredients (starch and milk proteins) and in processed puree at each processing step. Steam cooking of raw zucchinis and pasteurization of puree in the final package significantly reduced spore numbers to 0.5+/-0.3 log CFU/g in the processed food. During storage, numbers of spore-forming bacteria increased up to 7.8+/-0.1 log CFU/g in puree after 5 days at 20-25 degrees C, 7.5+/-0.3 log CFU/g after 21 days at 10 degrees C and 3.8+/-1.1 log CFU/g after 21 days at 4 degrees C. B. cereus counts reached 6.4+/-0.5 log CFU/g at 20-25 degrees C, 4.6+/-1.9 log CFU/g at 10 degrees C, and remained below the detection threshold (1.7 log CFU/g) at 4 degrees C. Our findings indicate that raw vegetables and texturing agents such as milk proteins and starch, in spite of their low levels of contamination with bacterial spores and the heat treatments they undergo, may significantly contribute to the final contamination of cooked chilled foods. This contamination resulted in growth of B. cereus and psychrotrophic bacterial spores during storage of vegetable puree. Ways to eliminate such contamination in the processing line are discussed.

SUMMARY Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

International audience; Sixteen gram-positive endospore-forming bacteria previously isolated from soil, plant rhizospheres, plant roots and pasteurized pureed vegetables were studied to determine their taxonomic positions. The isolates were formerly identified as Bacillus circulans based on their biochemical characters using API galleries. Two of these strains, RSA19T and TOD45T, were recently assigned to the genus Paenibacillus based on phylogenetic analysis of their 16S rRNA (rrs) gene sequence. In the present work, the sixteen isolates were assigned to two genomospecies using DNA-DNA hybridization, in agreement with rrs gene sequence analysis. These genomospecies can also be differentiated on the basis of their cultural and biochemical characters into two novel species, for which the names Paenibacillus graminis sp. nov. (type strain RSA19T = ATCC BAA-95T = LMG 19080T) and Paenibacillus odorifer sp. nov. (type strain TOD45T = ATCC BAA-93T = LMG 19079T) are proposed.

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25°C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis . A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans . Storage conditions at 4°C favored the development of “ B. macroides/B. maroccanus ” and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25°C favored the development of B. subtilis group ( B. licheniformis and B. subtilis ) and B. cereus group strains. At 10°C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus , and B. pumilus varied with the experiment.

In commercial purees of broccoli, carrot, courgette, leek, potato and split pea, pasteurized in their final packaging and analysed at two periods, Bacillus spp. were the dominant aerobic mesophilic bacteria (AMB). Initial numbers were generally lower than 2 log cfu g−1. They increased up to 6–8 log cfu g−1after about 20 days of storage at 10°C. At 4°C, numbers of AMB after 20 days were lower than 3 log cfu g−1in potato puree, lower than 4 log cfu g−1in leek puree, and between 3 and 6 log cfu g−1in other products. Strict anaerobes were in markedly lower numbers than AMB. At all storage temperatures tested courgette puree usually showed the most rapid bacterial growth and spoilage. On this product, an increase in storage temperature from 4°C to 10°C resulted in a threefold reduction in time to 5 log cfu g−1, and time to spoilage. Growth kinetics of AMB in courgette puree at 20°C, 15°C, 10°C, 6·5°C and 4°C were determined using a mathematical model. Three hundred and forty eight isolates were identified using the API system. Bacillus circulans, B. macerans and B. polymyxa were among the main species isolated from products stored at 4°C and 10°C, while B. subtilis and B. licheniformis were the dominant species in product stored at abuse temperature. Bacillus cereus was isolated from all storage conditions, but mostly from products stored at abuse temperature.

Biological control of foliar and post-harvest diseases, Wageningen, NLD, 1992/11/29-1992/12/04; International audience

Biological control of foliar and post-harvest diseases, Wageningen, NLD, 1992/11/29-1992/12/04; International audience