Kitchen layouts and consumers' food hygiene practices: Ergonomics versus safety.

Our paper emphasizes the importance of the kitchen layout in facilitating consumers' food hygiene practices. A significant correlation was found between the sink placement (inside or outside the kitchen) and hygienic practices during food handling based on a survey performed on consumers from ten European countries, indicating that those who had the sink in the kitchen were more likely to perform proper hygiene practices than those who have not. The self-reported practices were supported by observed practices in 64 households from five European countries. The observational study combined with the examination of kitchen layouts revealed that the kitchen work triangle with its apexes represented by the kitchen sink, cooking stove and refrigerator, which is recommended for ergonomic reasons by architects and designers, did not necessarily support food hygiene practices in kitchens. Cross-contamination events were associated with the sink - countertop distances longer than 1 m. Based on this, a new kitchen triangle with its apexes represented by the kitchen sink, working place (usually countertop) and cooking stove, with the distance between the sink and the working place less than 1 m is proposed to be used as norm in kitchen designs for combining ergonomics with safety. This triangle is proposedly named the food safety triangle and is aimed to mitigate the risks of foodborne illnesses by creating an arrangement that facilitates hygiene practices. This study is the first to highlight the importance of implementing the concept of food safety in the kitchen design based on significant correlations between kitchen equipment placement and consumers' food safety practices.

Data on European kitchen layouts belonging to vulnerable consumers (elderly people and young families with children or pregnant women) and risk-takers (young single men).

The data presented here capture the structure of kitchen layouts belonging to consumers vulnerable to foodborne diseases and food risk-takers. Data were collected in the frame of the SafeConsume project by multidisciplinary research teams that visited consumers during preparing a meal and had the possibility to examine their cooking routines. Distances between sink and stove, sink and refrigerator, stove and refrigerator, sink and working place (countertop or table), stove and working place were analyzed to correlate food safety practices applied during cooking with kitchen arrangements. The results arising from analyzing the ergonomics of kitchens versus potential cross-contamination events are presented in Mihalache et al., [1]. These data contribute to a better understanding of real kitchen layouts and can be used as a starting point for future research regarding food safety-oriented arrangements instead of ergonomics-focused designs, for food safety risk assessments, as study cases for explaining specific measures that can be established to improve food handling and hygiene practices in homes and for sociological research pointing consumers' behavior during cooking.

Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6-7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Consumer practices and prevalence of Campylobacter, Salmonella and norovirus in kitchens from six European countries.

About 40% of foodborne infections are acquired in the home. The aim of the present study was to track contamination of pathogens during domestic food preparation and link the contamination to preparation practices. Research participants from 87 households in six European countries were observed and interviewed during shopping and preparation of a chicken and vegetable meal. The presence of Salmonella spp., Campylobacter spp. and norovirus on raw chicken, kitchen surfaces, cloths and sponges was determined. The prevalence of Campylobacter on raw chicken varied from 8.3% in Norway (NO) to 80% in France (FR) and Portugal (PT), with a mean prevalence of 57%. Campylobacter was found on half of the products that had been frozen and appeared to be less prevalent on chicken from supermarkets than other sources. Salmonella was found in 8.6% of raw chicken samples, exclusively from Hungary (HU). A relationship between observed practices and spread of pathogens to kitchen surfaces was found only for the use of cutting boards for chicken and/or vegetables. After food preparation, Campylobacter and Salmonella were isolated from 23% (samples derived from HU, RO, UK) and 8.7% (HU), respectively of cutting boards. Research participants in France and Portugal were more likely to buy products that fitted their recipe, with less need for using cutting boards. Using the same board and knife for vegetables after using it for chicken and without washing with detergent was common in Portugal and Romania, but not in the other countries. Contamination with Campylobacter to other kitchen surfaces or washing utensils were found in five households (UK, RO, PT). Rinsing chicken in sinks was common in three countries (PT, HU, RO), and washing vegetables in the same sink was also usual. Prevalence of Norovirus was low, with detection in one out of 451 samples. The participants' awareness of the risk posed by pathogens from raw chicken differed among the six countries, with higher awareness in Norway and the UK than the other countries studied. In conclusion, practices intended to avoid cross-contamination from chicken to kitchen surfaces and washing utensils are not established among consumers in all European countries. Nevertheless, cross-contamination events that disseminate infectious doses of pathogens seems to be rare, probably due to the relatively low levels of pathogens in food combined with food preferences. Food safety interventions must consider the national food culture, preferences, practices and the prevalence and levels of pathogens in food. Emphasis should be on providing and promoting chicken products with lower risk (prevalence of pathogens, ready-to-cook) and safe use of cutting boards.

Impact of temperature and oxygen on the fate of Bacillus weihenstephanensis in a food-based medium.

The capacity of the Bacillus weihenstephanensis KBAB4 strain, a psychrotolerant species of the B. cereus sensu lato group, to multiply in carrot broth at 8 °C and 30 °C, in presence or absence of oxygen was determined. In aerobic carrot broth tyndallized in presence of oxygen, at both temperatures, the population of vegetative cells of B. weihenstephanensis inoculated at a level of 103 or 106 CFU/ml dropped immediately. After 16 h at 30 °C, B. weihenstephanensis reached around 103 CFU/ml, indicating that some vegetative cells had survived and multiplied, with lipid inclusions accumulated in cells, indicating possible stressing conditions. At 8 °C, no multiplication of B. weihenstephanensis was observed during 3 days to at least 12 days, depending of carrot broth batches. In anaerobic carrot broth tyndallized without oxygen, the vegetative cells of B. weihenstephanensis were not killed upon inoculation and multiplied in the broth at both 30 °C and 8 °C. Comparison with results from previous studies shows that B. weihenstephanensis behaves differently in carrot broth and in laboratory media at 8 °C with regards to presence or absence of oxygen.

Bacillus cereus cshA Is Expressed during the Lag Phase of Growth and Serves as a Potential Marker of Early Adaptation to Low Temperature and pH.

Bacterial adaptation is characterized by a lag phase during which cells do not multiply or modify their physiology to cope with the constraints of their environment. Our aim was to determine a sequence of events during the lag phase of growth at low temperature and pH for three Bacillus cereus strains. The onsets of expression of two genes, one of which is essential for stress adaptation (cshA, coding for a RNA helicase) and one of which is involved in the transition between lag phase and exponential phase (abrB, coding for a transition regulator), were determined using fluorescent transcriptional reporter systems. Regardless of the stressing conditions and the tested strains, the cshA promoter was active very early, while the biomass increased and always did so before the first cell division. At 12°C and pH 7.0, the onset of cshA promoter activity occurred at between 3 h and 7 h, while the bacterial counts started to increase at between 12 h and 13 h. At pH 5.0 and at 20°C or 30°C, the onset of cshA promoter activity occurred before 1 h and earlier than at pH 7.0. In contrast, the onset of abrB promoter activity depended on the strain and the stressing conditions. In the ATCC 14579 strain, the onset of abrB promoter activity always started at between 30 min and 3 h, before biomass increased and cell division occurred. For the other strains, it took place along with the first cell division at 12°C but did so much later during growth under the other tested conditions.IMPORTANCE The spore-forming bacterium B. cereus is a major cause of foodborne outbreaks in Europe. Some B. cereus strains can grow at low temperatures and low pH in many processed foods. Modeling of the bacterial lag time is hampered by a lack of knowledge of the timing of events occurring during this phase. In this context, the identification of lag phase markers, not currently available, could be a real advance for the better prediction of lag time duration. Currently, no molecular markers of this phase are available. By determining that cshA was always expressed early during the lag phase, we provide a molecular marker of the early adaptation process of B. cereus cells when exposed to low temperature and pH.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Heat-resistance of psychrotolerant Bacillus cereus vegetative cells.

Spores of psychrotolerant strains of the foodborne pathogen Bacillus cereus can multiply during storage of cooked or pasteurized, refrigerated foods and can represent a risk if these cells are not eliminated during reheating of food product before consumption. We determined the heat-resistance of psychrotolerant B. cereus vegetative cells at different heating temperatures in laboratory medium and compared it with that of thermotolerant B. cereus vegetative cells. The z values, based on times for a 3 log10 reduction, of the vegetative cells of the three psychrotolerant phylogenetic groups of B. cereus varied between 3.02 °C and 4.84 °C. The temperature at which a 3 log10 reduction was achieved in 10 min varied between 47.6 °C and 49.2 °C for psychrotolerant vegetative cells and it was around 54.8 °C for thermotolerant vegetative cells. Moreover, 0.4 min at 60 °C would be sufficient for a 6 log10 CFU/ml reduction of the most heat resistant psychrotolerant B. cereus vegetative cells. These data clearly showed that psychrotolerant B. cereus vegetative cells can be rapidly eliminated by a mild heat treatment such as food reheating.

Combined effect of anaerobiosis, low pH and cold temperatures on the growth capacities of psychrotrophic Bacillus cereus.

Psychrotrophic strains of the foodborne pathogen Bacillus cereus can multiply during the refrigerated storage of food products. The aim of this study was to determine the impact of anaerobiosis on the growth of two psychrotrophic B. cereus strains exposed to acidic pH at a cold temperature in a laboratory medium. At 10 °C, growth occurred at pH values equal to or higher than 5.7 during anaerobiosis, whereas aerobic growth was observed from pH 5.4. Growth rates during aerobiosis were similar at pH 5.4 and pH 7. No growth was observed for the two tested strains at 8 °C without oxygen regardless of the pH; however, both strains grew at this temperature from pH 5.4 in the presence of oxygen. These pH growth limits in aerobiosis are consistent with those reported for different strains and different foods or media, but no other studies have described anaerobic growth at acidic pH values. The maximal B. cereus concentration was approximately 6.0 log10 CFU/ml for cultures in the absence of oxygen and approximately 8.0 log10 CFU/ml for cultures in the presence of oxygen. In conclusion, we found that the combination of anaerobiosis, pH < 5.7 at 10 °C, or anaerobiosis and temperatures ≤8 °C prevent psychrotrophic B. cereus growth.

Agrifood systems and the microbial safety of fresh produce: Trade-offs in the wake of increased sustainability.

Fresh produce has been a growing cause of food borne outbreaks world-wide prompting the need for safer production practices. Yet fresh produce agrifood systems are diverse and under constraints for more sustainability. We analyze how measures taken to guarantee safety interact with other objectives for sustainability, in light of the diversity of fresh produce agrifood systems. The review is based on the publications at the interface between fresh produce safety and sustainability, with sustainability defined by low environmental impacts, food and nutrition security and healthy life. The paths for more sustainable fresh produce are diverse. They include an increased use of ecosystem services to e.g. favor predators of pests, or to reduce impact of floods, to reduce soil erosion, or to purify run-off waters. In contrast, they also include production systems isolated from the environment. From a socio-economical view, sustainability may imply maintaining small tenures with a higher risk of pathogen contamination. We analyzed the consequences for produce safety by focusing on risks of contamination by water, soil, environment and live stocks. Climate change may increase the constraints and recent knowledge on interactions between produce and human pathogens may bring new solutions. Existing technologies may suffice to resolve some conflicts between ensuring safety of fresh produce and moving towards more sustainability. However, socio-economic constraints of some agri-food systems may prevent their implementation. In addition, current strategies to preserve produce safety are not adapted to systems relying on ecological principles and knowledge is lacking to develop the new risk management approaches that would be needed.

Identification of Fatty Acids in Bacillus cereus.

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species. Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs. Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs. The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Role of fatty acids in Bacillus environmental adaptation.

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.

Fatty acid profiles and desaturase-encoding genes are different in thermo- and psychrotolerant strains of the Bacillus cereus Group.

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.

The adaptive response of bacterial food-borne pathogens in the environment, host and food: Implications for food safety.

Bacteria are constantly faced to stress situations in their ecological niches, the food and the host gastrointestinal tract. The capacity to detect and respond to surrounding changes is crucial for bacterial pathogens to survive or grow in changing environments. To this purpose, cells have evolved various sophisticated networks designed to protect against stressors or repair damage caused by them. Challenges can occur during production of foods when subjected to processing, and after food ingestion when confronted with host defensive barriers. Some pathogenic bacteria have shown the capacity to develop stable resistance against extreme conditions within a defined genomic context and a limited number of generations. On the other hand, bacteria can also respond to adverse conditions in a transient manner, through the so-called stress tolerance responses. Bacterial stress tolerance responses include both structural and physiological modifications in the cell and are mediated by complex genetic regulatory machinery. Major aspects in the adaptive response are the sensing mechanisms, the characterization of cell defensive systems, such as the operation of regulatory proteins (e.g. RpoS), the induction of homeostatic and repair systems, the synthesis of shock response proteins, and the modifications of cell membranes, particularly in their fatty acid composition and physical properties. This article reviews certain strategies used by food-borne bacteria to respond to particular stresses (acid, cold stress, extreme pressure) in a permanent or transient manner and discusses the implications that such adaptive responses pose for food safety.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available.

The CasKR two-component system is required for the growth of mesophilic and psychrotolerant Bacillus cereus strains at low temperatures.

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 (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at Tmin. 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.

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature.

Inferring an augmented Bayesian network to confront a complex quantitative microbial risk assessment model with durability studies: application to Bacillus cereus on a courgette purée production chain.

The Monte Carlo (MC) simulation approach is traditionally used in food safety risk assessment to study quantitative microbial risk assessment (QMRA) models. When experimental data are available, performing Bayesian inference is a good alternative approach that allows backward calculation in a stochastic QMRA model to update the experts' knowledge about the microbial dynamics of a given food-borne pathogen. In this article, we propose a complex example where Bayesian inference is applied to a high-dimensional second-order QMRA model. The case study is a farm-to-fork QMRA model considering genetic diversity of Bacillus cereus in a cooked, pasteurized, and chilled courgette purée. Experimental data are Bacillus cereus concentrations measured in packages of courgette purées stored at different time-temperature profiles after pasteurization. To perform a Bayesian inference, we first built an augmented Bayesian network by linking a second-order QMRA model to the available contamination data. We then ran a Markov chain Monte Carlo (MCMC) algorithm to update all the unknown concentrations and unknown quantities of the augmented model. About 25% of the prior beliefs are strongly updated, leading to a reduction in uncertainty. Some updates interestingly question the QMRA model.

Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning.

An aerobic endospore-forming bacillus (NVH 391-98(T)) 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. nov. is proposed, with the type strain NVH 391-98(T) (= DSM 22905(T) = CIP 110041(T)).