Adaptation of Bacillus cereus, an ubiquitous worldwide-distributed foodborne pathogen, to a changing environment

Food Research International 43 (2010) 1885–1894

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Adaptation of Bacillus cereus, an ubiquitous worldwide-distributed foodborne pathogen, to a changing environment Frédéric Carlin a,b,*, Julien Brillard a,b, Véronique Broussolle a,b, Thierry Clavel a,b, Catherine Duport a,b, Michel Jobin a,b, Marie-Hélène Guinebretière a,b, Sandrine Auger c, Alexei Sorokine c, Christophe Nguyen-Thé a,b a b c

INRA, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon F-84000, France Université d’Avignon et des Pays de Vaucluse, UMR408 Sécurité et Qualité des Produits d’Origine Végétale, Avignon F-84000, France INRA, UR895 Génétique Microbienne, F-78000 Jouy-en-Josas, France

a r t i c l e

i n f o

Article history: Received 2 June 2009 Accepted 25 October 2009

Keywords: Bacillus cereus Genetic Spore Adaptation Environment Food Food processing Emergence Climate change

a b s t r a c t 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. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction An increase in mean temperatures and a change in precipitation regimes are likely the main scientific evidence of climate change in recent decades (Trenberth et al., 2007). Climate change modifies the natural habitat of living organisms, as already observed for several species of animals or plants (Root et al., 2003; Rosenzweig et al., 2008). Gale, Drew, Phipps, David, and Wooldridge (2009) suggest that the prevalence and occurrence of livestock diseases in Great Britain will continue to be affected by climate change for different reasons including changes in vector ecology, farming practices or environmental factors such as temperature or humidity. Global warming may be also a concern for public health, including diseases transmitted by foods. An increase in diarrheal diseases caused by foods such as salmonellosis is suggested,

* Corresponding author. Address: UMR 408 INRA-UAPV Sécurité et Qualité des Produits d’Origine Végétale, Site Agroparc, 84914 Avignon Cedex 9, France. Tel.: +33 (0) 4 32 72 25 19; fax: +33 (0) 4 32 72 24 92. E-mail address: [email protected] (F. Carlin). 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.10.024

assuming that Salmonella growth is favoured by higher temperatures (McMichael, Woodruff, & Hales, 2006). The natural spread of foodborne pathogens may change, and their ecological niches may eventually extent. For instance a Vibrio parahaemolyticus outbreak caused by oysters has been reported in 2005 in Alaska, 1000 km to the north of the previously documented outbreak of the same origin. This unusual outbreak of foodborne poisoning has been attributed to a never attained Pacific Ocean temperature in the area of oyster farming (McLaughlin et al., 2005). Consequences of global warming on foodborne pathogens ecology, on their saprophytic life in soil or water, or on their commensal, symbiotic or parasitic life in animals are likely complex and difficult to forecast. However changes affecting microorganisms will likely involve already known evolution or adaptation mechanisms. What could be those adaptation mechanisms in a foodborne pathogen? Among foodborne pathogenic bacteria, Bacillus cereus is ubiquitous and worldwide distributed in the environment, displays a large variety of characters of adaptation and therefore represents an interesting model to study the potential impact of climate change on foodborne pathogenic bacteria. The aim to this presentation is to review some aspects of the B. cereus biology that may be

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relevant to this issue and to discuss what could make B. cereus affected (or not) by climate change. 2. General features of B. cereus The B. cereus sensu lato (or B. cereus group) displays a wide diversity of strains belonging to six closely related species: B. cereus sensu stricto and Bacillus anthracis, known as human pathogens, Bacillus thuringiensis used as biopesticide, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. From previous phylogenetic studies (Helgason, Tourasse, Meisal, Caugant, & Kolsto, 2004; Priest, Barker, Baillie, Holmes, & Maiden, 2004; Sorokin et al., 2006), a division of the B. cereus sensu lato phylogenetic structure into seven major phylogenetic groups (I–VII) by using both genetic and phenotypic criteria was recently proposed (Guinebretiere et al., 2008). The previously mentioned species were since distributed and labeled with a specific nomenclature (Table 1). In particular each of these seven major phylogenetic groups corresponds to a specific ‘‘thermotype”, showing clear differences in ability to grow at low or high temperatures. Different virulence potentials could also be associated with these genetic groups. In addition group VII strains in this phylogenetic scheme were suggested to be recognized as a novel species ‘‘Bacillus cytotoxicus” having a clearly distinguishing moderate thermotolerant phenotype (Auger et al., 2008; Guinebretiere et al., 2008; Lapidus et al., 2008). B. cereus spores and cells are common in soil, in water and in the gastro-intestinal tract of eukaryotic organisms (Jensen, Hansen, Eilenberg, & Mahillon, 2003; Swiecicka, 2008). This wide and ubiquitous distribution in the environment, together with the formation of endospores highly resistant to heat, chemical and other environmental stresses, obviously favor B. cereus contamination in foods. Psychrotolerant strains may represent the main concern during storage of refrigerated foods. Prevalence and/or concentrations in food raw material and in processed foods are extensively looked for and reported. For example a comprehensive review including data on B. cereus ecology, prevalence and growth capacity in foods, and resistance to process was published by the European Food Safety Agency (Anonymous, 2005). B. cereus is a common source of foodborne poisoning representing 1–33% of cases of foodborne poisonings depending on countries (Anonymous, 2005). B. cereus causes two types of syndromes of foodborne poisonings: emetic caused by a cyclic peptide called cereulide, and diarrheic, caused by several types of toxins (Table 2). Both syn-

dromes are usually mild, but a few fatal cases have been reported. Foods linked to B. cereus emetic or diarrheal foodborne poisoning are frequently heat-treated and/or not adequately refrigerated after preparation and before consumption. Rice and pasta dishes have frequently caused emetic poisonings (Anonymous, 2005). 3. Lessons from genetics B. cereus sensu lato bacteria are particularly interesting because their genetic background confers variable tolerance to temperature. Indeed, the global evolution of B. cereus sensu lato is not anarchic but seems to be strongly determined by ecological adaptations. As an illustration, the seven major phylogenetic groups (I–VII) share a particular ‘ecotype’ structure in which each phylogenetic group exhibits its proper range of growth temperature and is for this reason associated with particular thermal niches (Table 1) (Guinebretiere et al., 2008). This genetic diversification associated with modifications of temperature tolerance limits is a first example of the genetic adaptive faculty of B. cereus sensu lato. We may speculate that the emergence of more coldadapted populations or more warm-adapted populations corresponds to the colonization of new or different environments for which B. cereus organisms had to adapt. This ecological diversification can be genetically marked by the relative abundance of the ‘psychrotolerant’ rrs signatures (as defined by Pruss, Francis, von Stetten, & Scherer, 1999), and the cspA signature (as defined by Francis, Mayr, von Stetten, Stewart, & Scherer, 1998), as traces of particular different strategies set up by all groups to adapt to new environments and particularly to temperature changes (Guinebretiere et al., 2008). Thus the rich adaptive ability within B. cereus sensu lato permits the development of new evolutionary lines and long-term persistence of B. cereus towards climate change. Global warming may also signify homogenization of the actual populations towards a thermotolerant status. It is probably very indicative that many strains isolated from food poisoning cases have a tendency rather to be more thermotolerant. Only 10–15% of isolates of the most cold-adapted groups II, V and VI, but 35% of isolates of more thermotolerant groups III, IV and VII, are associated with outbreaks of foodborne poisonings (Guinebretiere et al., 2008). This tendency includes the very important cluster of the emetic strains (Carlin et al., 2006; Ehling-Schulz, Svensson, et al., 2005, Ehling-Schulz, Vukov, et al., 2005; Vassileva et al., 2007), which was also the cause of several fatal foodborne poisoning cases (Dierick et al., 2005; Mahler et al., 1997) and strains

Table 1 The seven Bacillus cereus genetic groups (Guinebretiere et al., 2008) and their main features. Group

Association to currently defined species

Association to cases of food poisoning

Domain of growth temperatures (°C)

Heat resistance of sporesa

Observations

I II

B. pseudomycoides B. cereus II, B. thuringiensis II Emetic strains B. cereus III, B. thuringiensis III B. anthracis B. cereus IV, B. thuringiensis IV B. cereus V, B. thuringiensis V B. weihenstephanensis B. mycoides B. thuringiensis VI ‘B. cytotoxicus’

No Yes

10–40 7–40

? ++

Yes

15–45

+++

Mesophilic; rhizoidal colonies on agar Most strains are psychrotolerant. ‘‘Psychrotolerant” cspA signature is absent Mesophilic

Yes

10–45

++

Mesophilic

Yes

8–40

++

Mesophilic

No

5–37

+

All strains are psychrotolerant, with a ‘‘psychrotolerant” cspA signature (Francis et al., 1998) specific of this group

Yes

20–50

+++

Thermotolerant; rare isolates from this group, but commonly associated to outbreaks of foodborne poisonings

III

IV V VI

VII a

Ranking established from Afchain et al. (2008) and Carlin et al. (2006).

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F. Carlin et al. / Food Research International 43 (2010) 1885–1894 Table 2 Main characteristics of foodborne poisonings caused by Bacillus cereus. Type of foodborne poisoning

Primary cause

Symptoms

Infectious dose

Toxins and genes

Emetic

Production of emetic toxin in food prior to ingestion Ingestion of cells and spores and toxin production in the small intestine

Outbreaks associated to foods containing >105 B. cereus/g 105–108 cells or spores, i.e. foods containing >104/g may pose a problem

Cereulide (cyclic peptide)/ces

Diarrheal

Vomiting 1–5 h after ingestion of the contaminated food, often followed by diarrhea 8–16 h after ingestion Watery diarrhea and abdominal pain 8–16 h after ingestion of the contaminated foods

Hemolysin BL. Toxin formed by three proteins (B, L1, L2), coded by three genes organized in 1 operon (hblA, hblD, hblC, respectively) Non-hemolytic entrotoxin (NHE). Toxin formed by three proteins (A, B, C), coded by three genes organized in 1 operon (nheA, nheB, nheC, respectively). Cytoxin K. One single protein coded bt CytK

Adapted from Anonymous (2005), Ehling-Schulz, Vukov, et al. (2005), Granum (2007), and Kramer and Gilbert (1989).

producing the anthrax toxins (group III). Even more pronounced is the correlation between ability to grow at elevated temperatures and the potential food toxicity for the group VII strains. Despite a small sample size due to rare isolation, four out of the five strains isolated at the time of writing have been associated with severe food poisonings (Fagerlund, Brillard, Furst, Guinebretiere & Granum, 2007; Lund, De Buyser, & Granum, 2000; Rau, Perz, Klittich, & Contzen, 2009; Guinebretière et al., unpublished data). The natural ecological niche of ‘‘B. cytotoxicus” group VII strains has not yet been elucidated. However their isolation from the four outbreaks of foodborne poisonings was always related to dehydrated vegetables, similarly to emetic strains. This suggests a selection of the more thermotolerant strains by heat during the drying process of dehydrated foods (Guinebretiere et al., 2008). Conversely group VI, with the lowest growth temperature range, has never been implicated in outbreaks of foodborne poisonings. Among B. cereus isolates from cooked chilled foods, those with the lowest minimal growth temperatures produced supernatant with the lowest toxicity to Caco 2 cells (Choma et al., 2000). Similarly, in dairy products and dairy farms, psychrotrophic isolates produced very low amounts of the enterotoxins Hbl and Nhe (Svensson, Monthan, Guinebretiere, Nguyen-The, & Christiansson, 2007). Toxicity to Caco 2 cells represent a global measure of the activity of B. cereus enterotoxins (Moravek et al., 2006) and high enterotoxin productions was identified as a feature of B. cereus strains involved in foodborne poisoning (Guinebretiere, Broussolle, & Nguyen-The, 2002). Although the genetic structure proposed by Guinebretiere et al. (2008) was not available when the above mentioned works on cooked chilled foods and dairy products were published, these psychrotrophic strains with a low toxicity presumably belonged to group VI. Although other psychrotrophic groups (i.e. group II) have been involved in foodborne diseases (Guinebretiere et al., 2008), it might be possible that a shift in B. cereus populations towards more thermophilic groups decreases the prevalence in foods of strains with the lowest association with virulence (i.e. strains from group VI), and increases the prevalence of the most hazardous B. cereus strains (i.e. mesophilic groups). The discovery of group VII strains is recent and their isolation is still a rare event, but interestingly associated with outbreaks of foodborne poisoning. This addresses to the understanding of their ecology and natural occurrence. Are such strains more frequently isolated in the recent few years because of improving of strain identification protocols (this is unlikely, as advances in B. cereus detection methods have not been so important in recent years) or is it due to the emergence of a real and new danger from this group of thermotolerant strains? Do they represent a real danger as a food emerging pathogen? Also an important practical question is what the real pathogenic capacities of such strains are and how

frequent are the toxic strains between others. The first isolate, NVH391-98, was reported as the cause of the fatal poisoning of three persons from 45 having severe necrotic diarrhea (Lund et al., 2000). It appears that the other isolates are less dangerous, but a systematic study using adequate model system is needed to clarify the question. The strain NVH391-98 (group VII) was revealed as very different by comparison of its genome with other strains of the B. cereus group (Lapidus et al., 2008). One of the striking finding from the genomic sequence was the absence of rB RNA polymerase subunit, one of the most important transcriptional regulator of stress response in sporulating Bacilli (see below). The stress response regulation should therefore be very different in the ‘‘B. cytotoxicus” strains and such strains represent a natural model to study responses that are rB independent. Another striking difference is that the size of the genome of this strain (4.1 Mb) is much smaller than that of other B. cereus sensu lato strains (5.2–5.4 Mb). Due to this reduced genome size the strain lacks many genes that are normally rather characteristic of other groups of B. cereus strains. This, in its turn, should reduce the abilities of such strains to be adapted to different environmental conditions and to different stresses. Similar significant modification of the genome size have been previously associated with a modification of habitat for Frankia sp. (Normand et al., 2007), the smallest genomes being related to specific/restricted niches (for example the host) and the largest to large environmental niches (such as soil). All these data indicate that the group VII, although highly virulent and thermotolerant, may be restricted to its specific but still unknown niche, whereas all other groups have not this handicap and may spread around, subject to set up appropriate adaptation strategies. Whatever the genetic possibilities are, it seems to be important to understand the real impact of mesophilic and moderately thermotolerant bacteria of B. cereus sensu lato on the human activity related to the food poisoning and the risk of emergence of such pathogens in relation to the global climate change.

4. Environment of sporulation and spore properties In Bacillus sp., the process leading to spore production from vegetative cells (sporulation) is triggered by nutrient depletion and quorum sensing (Piggot & Hilbert, 2004; Setlow & Johnson, 2007). Sporulation at frequencies of 50–90% is easy to obtain in the laboratory with most Bacillus sp. Laboratory experiments with the model bacterium Bacillus subtilis, with B. cereus and other spore-forming bacteria demonstrate that the environment of sporulation has a critical influence on spores. This may affect spore resistance to physical or chemical stresses applied in the food

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industry, spore structures and composition (spore dryness for instance), or spore germination that gives a vegetative cell able to multiply (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000) (Table 3). A 10-fold increase in the decimal reduction time of several B. cereus strains for an increase in sporulation temperature of 25 °C has been reported for instance (Leguerinel, Couvert, & Mafart, 2007). In B. subtilis, changes in the composition of spores or in their structural components (coats, cortex, soluble proteins (SASP) bound to DNA, etc.. . .) influence spore properties and germination ability, and characterization of spores formed in diverse environments has shown that these structures or composition may be influenced by the environment of sporulation (Nicholson et al., 2000). This is less studied in B. cereus; however it is reasonable to consider that it is not dramatically different. Consequently any climate change affecting the environment of sporulation may potentially induce a modification of spore properties. However those modifications will likely be difficult to detect. Considering temperature for instance, global warming will represent only a few °C increase in average temperature, while laboratory observations are obtained with higher temperature increases. What is the natural environment of sporulation of B. cereus? B. cereus life cycle (including germination, growth and sporulation) is commonly associated with soil and has been observed in liquid soil extract or in artificial soil microcosm (Vilain, Luo, Hildreth, & Brozel, 2006). B. cereus is also strongly associated with eukaryotic soil animals, colonizing the gut of invertebrates, and possibly being intestinal symbiots (Konig, 2006; Margulis et al., 1998). In earthworms, germination seems to be restricted to the gut and sporulation may occur after defecation (Hendriksen & Hansen, 2002; Swiecicka, 2008). Multiplication and sporulation seem to be also associated with death of their hosting animal, which may have been caused by the bacterium itself (B. thuringiensis for insects, B. anthracis in mammals), or in the case of B. cereus sensu stricto during co-infection of insects with B. thuringiensis (Raymond, Lijek, Griffiths, & Bonsall, 2008). Consequently this suggests that climate change may influence the viability of B. cereus in two aspects. Climate change may modify the physical and chemical environment of spores (change in soil temperature, humidity). As demonstrated with B. anthracis, intensive humidity after a long dry period is the most favorable condition for the illness spread (Gale et al., 2009; Nishi et al., 2002). Climate change may also modify the spreading of B. cereus hosts. These hosts are in general terms part of wildlife, mainly insects and other arthropods, or earthworms. Earthworms populations for instance might be affected by a temperature increase (Zaller et al., 2009), although in a complex way (Eggleton, Inward, Smith, Jones, & Sherlock, 2009). In addition, if B. cereus hosts are invertebrates, the bacterium might be more directly

affected by environment temperature than if it was living in the gut of warm blood animals. However recent work showed that B. thuringiensis was able to complete its life cycle in the intestine of germ-free rats (Wilcks et al., 2008). Whether this can also occur in rats with their normal intestinal flora is not known. At the present time there is no indication whether impact of climate change on soil fauna may affect B. cereus host range. B. cereus also seems to be resident in food industries in some cases: RAPD-fingerprinting of B. cereus from dairy farms and plants suggested the presence of an in-house microflora in some instances (Svensson, Ekelund, Ogura, & Christiansson, 2004). In such conditions sporulation will depend on the processing environment and not on climate. 5. Mechanisms of adaptation of B. cereus to environment changes The ability of B. cereus to survive (spores) and colonize (vegetative cells) highly diverse ecological niches might explain the important role played by this bacterium in food safety issues. Indeed, B. cereus is able to multiply from biotic or abiotic soil fractions (Vilain et al., 2006) to human gastro-intestinal tract, passing through foods environments. In addition, the opportunistic virulence properties of B. cereus, also able to cause various types of systemic and local infections in humans (i.e. endophtalmitis, wound infection, septicemia. . .) (Schoeni & Wong, 2005) is presumably a consequence of its ability to adapt to various hosts and environments. To face these changing and sometimes hostile environments, B. cereus has to develop an adaptive strategy, elaborating different physiological responses depending on the stress encountered. 5.1. Signal transduction mechanisms involved in adaptation to changing environments In bacteria, recognition and response to a variety of environmental stimuli necessitate various signal transduction mechanisms (Marles-Wright & Lewis, 2007). Several alternative sigma factors play key roles in regulating bacterial gene expression necessary to adapt to rapidly changing conditions (Chaturongakul, Raengpradub, Wiedmann, & Boor, 2008). Among them, extracytoplasmic function (ECF) sigma factors are used to signal extracytoplasmic conditions to the cytoplasm (Helmann, 2002). Analysis of gene sequence shows that several of these ECF are present in B. cereus, but their exact function remains to be described. Another alternative sigma factor, rB, is involved in stress–response of Gram-positive bacteria and has been well characterized in B. cereus

Table 3 Evidence for the influence of sporulation environment on properties of B. cereus sensu lato spores. Factor involved during sporulation

Induced spore properties

References

Temperature

Increased damage to exosporium at 40 °C

Temperature

Higher resistance to heat, acid, alkaline and hydrogen peroxide stress of spores formed at 45 °C Lower heat resistance and higher germination of spores formed at low temperatures D-values increase as sporulation temperature increase

Faille, Tauveron, Gentil-Lelievre, and Slomianny (2007) Baweja et al. (2008)

Temperature Temperature Temperature Phosphate concentration in nutrient agar Glutamate concentration in sporulation medium Surface water conditions and viscosity of nutrient agar

Higher heat resistance and better germination with high glutamate

Gounina-Allouane, Broussolle, and Carlin (2008) Gonzalez, Lopez, Martinez, Bernardo, and Gonzalez (1999) Raso, Gongora-Nieto, Barbosa Canovas, and Swanson (1998) Mazas, Fernandez, Alvarez, Lopez, and Bernardo, 2009 de Vries, Atmadja, et al. (2005)

Time to first decimal reduction (d) at 88 °C varied in the range 1–3 according to the sporulation medium

Stecchini, Spaziani, Del Torre, and Pacor (2009)

Spores formed at the lowest temperatures are the most resistant to initiation of germination and inactivation by HHP Phosphate addition increases heat resistance

F. Carlin et al. / Food Research International 43 (2010) 1885–1894

(van Schaik & Abee, 2005; van Schaik et al., 2007). rB is activated by a heat-shock for instance and deletion of the sigB gene leads to a higher heat-sensitivity of heat-shocked vegetative cells, to modifications of spore structure, to alterations in spore germination, and surprisingly, to hydrogen peroxide hyperresistance (de Vries, Atmadja, Hornstra, de Vos, & Abee, 2005; de Vries, Hornstra, et al., 2005; van Schaik, Tempelaars, Wouters, de Vos, & Abee, 2004; van Schaik, Zwietering, de Vos, & Abee, 2005). Similarly sigB null mutants in B. anthracis are less virulent than the parental strain (Fouet, Namy, & Lambert, 2000). In addition, two-component systems (TCS) are frequently involved in signal transduction mechanisms encountered during response to changing environments (Hoch, 2000). TCS are characterized by a sensor histidine kinase (HK), often located in the membrane and coupled with a cognate response regulator. Perception of a particular stimulus by the HK leads to its autophosphorylation. The phosphoryl group is then transferred to the receiver domain of the response regulator. This activation of the response regulator usually leads to transcriptional regulation (activation or repression) of genes due to its direct interaction with specific DNA targets (Fig. 1). TCS are widespread among bacteria, and such systems are able to sense changes of conditions as diverse as osmolarity, pH, temperature, lack of essential nutrients, or host interaction signals. Thus, some TCS are vital for bacterial virulence, biofilm formation, or antibiotic resistance. As an illustration, the TCS DesKR of B. subtilis is a thermosensor and regulator able to adapt the bacteria to a rapid decrease in temperature. Optimal membrane fluidity is required for physiological exchanges between the cytoplasm and the extracellular environment. The lowered membrane fluidity caused by the drop in temperature (i.e. after a shift from 37 °C to 15 °C) is sensed by the membrane domain of the DesK HK. DesK autophosphorylates and then activates the DesR response regulator. DesR is a transcriptional activator able to recognize the promoter region of the gene encoding a D5-desaturase. This enzyme introduces double bounds in fatty acid moieties already present in the membrane. Thus, activation of expression of the desaturase gene causes an increase in the level of unsaturated fatty acid in the membrane, which leads to a regained fluidity (Aguilar, Hernandez-Arriaga, Cybulski, Erazo, & de

Extracellular redox signal

ResE

wall

H2N

Intracellular redox signal

ResD ~P HOOC

cytosol

ResD~P

Redox-dependent gene regulation Fig. 1. An illustration of a two-component signal (TCS) transduction system of B. cereus: redox signal transduction by ResDE. The membrane-bound kinase sensor ResE autophosphorylates in response to an intracellular redox signal and possibly to an extracellular redox signal. The PAS domain (gray circle) situated in the linker region between the NH2 transmembrane and COOH cytosolic domains of ResE is probably required for sensing the intracellular redox conditions. The C-terminal transmitter domain (hatched square) of ResE consists of an ATP-binding domain and a so-called H box domain that includes the conserved His residue for selfphosphorylation. Subsequently, the His-bound phosphoryl group of ResE is transferred onto a specific Asp residue in the cytosolic response regulator ResD. Phosphorylation of ResD alters its ability to interact with either DNA, or DNA and RNA polymerase, and thus to activate or repress transcription of a set of genes, which respond to redox signals.

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Mendoza, 2001; Schumann, 2009). The number of TCS encoding genes found in a bacterium is usually proportional to the size of its genome (Alm, Huang, & Arkin, 2006; Galperin, 2005). Genome sequencing of B. cereus strain ATCC14579 has revealed the presence of 55 sensor kinases (Anderson et al., 2005; Ivanova et al., 2003). Orthologs of most of them are found in members of the other B. cereus groups for which the genome sequence is available, including B. anthracis and B. thuringiensis (de Been, Francke, Moezelaar, Abee, & Siezen, 2006). In B. cereus sensu lato bacteria,, only very few of them have been characterized. An in silico approach has recently been proposed to predict the response regulator target genes and therefore to provide insight in the role of some TCS (de Been, Bart, Abee, Siezen, & Francke, 2008). Up to now, some of the B. cereus TCS have been shown to play a role in bacterial adaptation to its environment. The ResDE TCS is conserved in many bacteria, including B. cereus, and is involved in adaptation to low oxidation–reduction potential (Duport, Zigha, Rosenfeld, & Schmitt, 2006). Another TCS necessary for optimal growth at low temperature was recently identified in B. cereus (Brillard, unpublished data) and determining whether this TCS functions similarly to the B. subtilis TCS DesKR is an interesting issue. TCS controlling expression of virulence factors have been shown in a wide range of pathogenic bacteria (Stephenson & Hoch, 2004). In B. cereus, the above mentioned ResDE TCS was shown to control enterotoxin production (Hbl and Nhe) under anaerobiosis (Duport et al., 2006). Similarly, the B. cereus YvfTU TCS is involved in expression of a transcriptional activator (PlcR), a major virulence factors regulator. In the YvfTU mutant, a reduced virulence was observed using an insect model (Brillard et al., 2008). Consequently bacteria such as B. cereus, which encodes numerous TCS, likely adapt to environmental (climate) changes, and therefore better survive and eventually colonize locally and at least temporarily new niches. The processes that will lead to the occurrence of outbreaks of food poisonings are complex, but such adaptation might contribute at the end of the chain to a progressive change in prevalence or concentrations of B. cereus in foods. 5.2. Mechanisms of adaptation to some changing environments 5.2.1. Temperature adaptation As previously detailed the growth domains of the B. cereus strains range from nearly thermophilic to psychrotrophic strains, with a strong correlation with recently described phylogenetic clusters (Guinebretiere et al., 2008). Whatever their growth temperature limits, bacteria have to adapt to temperature changes. Mechanisms of adaptation to their lower temperature or higher temperature growth limits show substantial differences. Cold is a physical stress that drastically modifies all physical and chemical parameters of the bacteria, which subsequently respond in two phases, a cold-shock response and a continuous acclimatization response. Following cold-shock, there is a transient inhibition in the synthesis of most cellular proteins causing lack of cell growth, and a synthesis of cold-shock proteins (CSP). Five CSPs have been described in B. cereus with two of them highly synthesized upon cold-shock (Mayr, Kaplan, Lechner, & Scherer, 1996). Similarly to other species, B. cereus CspA and CspE likely act as RNA chaperones interacting with folded mRNA at low temperature. It should be noticed that cspA and 16S rDNA sequences of psychrotolerant B. cereus are clearly different of the sequences of mesophilic strains (Lechner et al., 1998) and may therefore constitute signatures of a psychrotolerant character. The exact function of cspA signatures in the growth potential of psychrotolerant B. cereus at low temperature is not clearly stated. They could contribute to the existence of ribosomes able to translate at low temperature mRNA into proteins. Besides the transient CSPs proteins, synthesis of some cold-acclimation proteins or CAPs occurred upon shift tem-

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perature and continues even when cold-stressed cells resume multiplication. These CAPs included proteins involved in cell metabolism, DNA and RNA metabolism, in protein folding and degradation, and in gene expression or protein synthesis. Previously described in Escherichia coli, their presence was evidenced in a transcriptomic and proteomic analysis of B. subtilis downshift from 37 °C to 15 °C (Budde, Steil, Scharf, Volker, & Bremer, 2006). Growth at low temperature is slower and hence all metabolic activities are adjusted to the new growth rate. In psychrotrophic B. cereus strains, glucose is metabolized with an increased participation at low temperature of the pentose–phosphate pathway (PPP) (Choma et al., 2000; Chung, Cannon, & Smith, 1976). A similar result was obtained with the mesophilic B. cereus ATCC14579, as a mutant for one of the PPP-enzyme encoding gene exhibits a strong growth defect at low temperature (Broussolle, unpublished data). Change in membrane fluidity is among the best characterized physiological consequences of cold-shock in bacteria. As temperature drops down, cell membrane changes from a liquid fluid crystalline to a gel rigid state. B. cereus adapts membrane fluidity at lower growth temperature by decreasing the proportion of branched-chain fatty acids and their equivalent chain-length, and increasing the anteiso-/iso-branched ratio and proportion of unsaturated fatty acids (Haque & Russell, 2004). Whether this membrane adaptation in B. cereus ATCC14579 is controlled by a twocomponent system, as described for the DesKR system of B. subtilis (Aguilar et al., 2001), remains to be determined. Sigma factors allow bacterial cells to regulate gene expression in response to environmental variations including variations in temperature. The heat stress response of B. cereus involves this regulator as sigB null mutant cells were less resistant to high temperature exposure than WT cells (van Schaik et al., 2004). Concerning low temperature adaptation, the role of rB has been demonstrated in growth and survival at refrigeration temperature of the foodborne pathogen Listeria monocytogenes (Becker, Evans, Hutkins, & Benson, 2000) and for B. subtilis (Brigulla et al., 2003). A cold-sensitive phenotype of B. cereus rB mutants has not been reported yet, whereas those mutants show adaptation defects to heat-shock, osmotic upshock or ethanol exposure (van Schaik et al., 2004). The rL factor was shown to play a role in cold-shock adaptation of B. subtilis membrane by regulation of the isoleucine metabolism implicated in the biosynthesis of the branched-chain fatty acid (Wiegeshoff, Beckering, Debarbouille, & Marahiel, 2006). Seeking after a specific r-factor involved in low temperature adaptation or cold-shock response of B. cereus strains is still a stimulating field of investigation. Long-term adaptation of B. cereus cells to high temperature is less extensively studied than its cold adaptation. The current understanding of heat stress response includes increased synthesis of a set of conserved heat-shock proteins (HSPs) such as the molecular chaperones DnaK, GroEL, and their cohorts of ATP-dependent proteases such as ClpP (Periago, van Schaik, Abee, & Wouters, 2002). These proteins play roles in protein folding, assembly, and repair and prevention of aggregation under stress conditions. The chaperones and proteases act together to maintain quality control of cellular proteins, as already described for a wide variety of bacteria. Cross-adaptations to different stresses was evidenced in both psychrotrophic and mesophilic strains of B. cereus (Periago, Abee, & Wouters, 2002; Periago, van Schaik, et al., 2002): exposure at low temperature has a benefit effect on cell survival at 50 °C and a weak induction of heat-shock proteins such as GroEL, DnaJ or DnaK chaperones was observed upon downshift temperature from 30 °C to 7 °C. A recent transcriptomic study demonstrate the higher resistance to heat of B. cereus cells exposed to salt stress (den Besten, Mols, Moezelaar, Zwietering, & Abee, 2009). All these results

suggest possible cross protection mechanisms for stress adaptation of B. cereus. 5.2.2. Carbohydrate (carbon source) and redox adaptation B. cereus possesses distinct catabolic routes allowing an efficient metabolism for energy from various carbohydrates and/or proteinaceous substrates under a wide range of redox conditions. This metabolic adaptation results in optimized growth (with the highest possible growth rate), even in absence of optimal growth conditions, in environments with available nutrients (soil, gastrointestinal tract of animals including man or foods) (Ivanova et al., 2003; Mols & Abee, 2008; Te Giffel, Beumer, Granum, & Rombouts, 1997). Efficient selection of the preferred carbon and energy source is critical for rapid growth and consequently B. cereus colonization is largely dependent of this adaptation. In environments that provide the cells with external electron acceptors such as oxygen (in aerobic environments in other words), reducing equivalents generated by glycolysis and Krebs cycle are re-oxidized by the respiratory chain, resulting in the buildup of a proton motive force and the subsequent synthesis of ATP by ATP synthase. In the absence of O2 (in anaerobic environments) or of other external electron acceptor (such as nitrate), ATP synthesis occurs at the level of substrate phosphorylation and the demand for redox neutrality is met by electron transfer from reducing equivalents (such as NADH) to an internal electron acceptor (such as pyruvate or acetyl coenzyme A). The latter results in a mixed acid fermentation of B. cereus with lactate, acetate, formate, ethanol, succinate, and traces of 2,3 butanediol as its typical products (Duport, Thomassin, Bourel, & Schmitt, 2004; Ouhib, Clavel, & Schmitt, 2006; Ouhib-Jacobs, Lindley, Schmitt, & Clavel, 2009; Rosenfeld, Duport, Zigha, & Schmitt, 2005; Zigha, Rosenfeld, Schmitt, & Duport, 2006). The abundance of genes encoding proteolytic enzymes and amino acid transporters (Ivanova et al., 2003), and amino acid utilization in growth and sporulation (de Vries, Atmadja, et al., 2005, de Vries, Hornstra, et al., 2005; Mols, de Been, Zwietering, Moezelaar, & Abee, 2007; Rosenfeld et al., 2005) also reveal a B. cereus amino acid preference. However, in anaerobic environments, B. cereus needs for proper growth carbohydrates (such as glucose, fructose or sucrose) in addition to amino acids (Duport et al., 2004; Ouhib et al., 2006; Ouhib-Jacobs et al., 2009). This adaptation to the nutrient source also modulates the production of B. cereus enterotoxins, which are major virulence factors. B. cereus Hbl enterotoxin production is lower during anaerobic growth on glucose than on fructose or sucrose (Duport et al., 2004; Ouhib et al., 2006; Ouhib-Jacobs et al., 2009). These results illustrate that, when grown anaerobically, the behavior of the foodborne pathogen B. cereus is extremely dependent upon the carbohydrate present in the medium. Furthermore, the modification of metabolic activity is reflected in the expression of the pathogenicity of the bacteria, notably its capacity to synthesize enterotoxins. This metabolic adaptation, occurring as different rates of formation of fermentation products, directly depends on redox of the growth medium (Duport et al., 2006). The ResDE TCS and the protein Fnr are two major redox controlling factors of catabolic pathways (Duport et al., 2006; Zigha, Rosenfeld, Schmitt, & Duport, 2007). ResDE consists of a membrane-bound histidine sensor kinase (ResE) and a cytoplasmic response regulator (ResD) (Fig. 1). ResE perceives signals related to oxidoreduction potential and undergoes autophosphorylation at a conserved histidine residue. The activity of ResD as a transcriptional activator is modulated by the level of phosphorylation, which could be inversely proportional to extracellular ORP (Esbelin, Armengaud, Zigha, & Duport, 2009). The redox signal activating ResE has not been identified but probably involves small effector molecules interacting with the PAS subdomain (Taylor & Zhulin, 1999) and possibly with the extracytoplasmic region (Baruah, Lindsey, Zhu, & Nakano, 2004).

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As in B. subtilis Fnr, B. cereus Fnr contains a C-terminal extension with four cysteine residues considered, in B. subtilis, to coordinate a [4Fe–4S]+2 center that serves directly as a redox sensor (Reents et al., 2006). However, unlike its ortholog in B. subtilis, B. cereus Fnr is able to function as a transcriptional factor independently of the integrity of the FeS cluster and thus exists in a active state whatever the extracellular ORP (Esbelin, Jouanneau, Armengaud, & Duport, 2008). These could illustrate the great redox adaptability of B. cereus compared to B. subtilis, the capacity of B. cereus group members to easily adapt to various redox conditions, and the coordination through the ResDE system and Fnr of metabolic adaptation and virulence gene expression in low redox environments such as animal gastro-intestinal tract (Moriarty-Craige & Jones, 2004). 5.2.3. Adaptation to low pH Adaptation to low pH environments plays a major role in survival of foodborne pathogens, as most natural environments or foods are not at optimal pH for B. cereus growth (7.0–7.5), or because of exposure to gastric acidity after ingestion by animals. Global change can modify pH of large environments. An ‘‘ocean acidification” has been observed and a decrease in a few tenths of pH units of surface ocean is predicted (Jacobson, 2005). Such an acidification in the usual B. cereus environment, soil for instance, has not yet been reported to our knowledge. Anyway it would probably be much more subtle than experimental pH changes. Acid environment leads to a reduction in cytoplasmic pH (pHi). The consequences of pHi reduction are a loss of activity of acid-sensitive enzymes (such as glycolytic enzymes) and structural damages to cell membranes and macromolecules such as DNA and proteins. Bacterial responses to low pH environments are a combination of constitutive and inducible strategies (Cotter & Hill, 2003). B. cereus vegetative cells, like many other bacteria, adapt to low pH through the induction of an Acid Tolerance Response (ATR) (Davis, Coote & O’Byrne, 1996; Jobin, Clavel, Carlin, & Schmitt, 2002; Ohara & Glenn, 1994; Thomassin, Jobin, & Schmitt, 2006; Tiwari, Sachdeva, Hoondal, & Grewal, 2004). Both psychrotrophic B. cereus TZ415 and mesophilic B. cereus ATCC14579 are more tolerant to acid shock when cells are beforehand cultivated at low pH (Browne & Dowds, 2002; Jobin et al., 2002; Thomassin et al., 2006). The B. cereus adaptation to acidity depends on various factors such as pH of prior growth or physiological state (Fig. 2) (Jobin et al., 2002; Thomassin et al., 2006). The acid survival of B. cereus requires protein neo-synthesis and the capacity of cells to maintain

Time (min) at pH 4.0 0

10

20

30

-1

log (N/N0 )

-2 -3 -4 -5 -6 Fig. 2. Acid Tolerance Response of B. cereus. Population reduction of steady-state B. cereus ATCC14579 cells grown at a pHo of 9.0 (), 7.0 (N), 6.0 (j) or 5.5 () and subjected to acid shock at pH 4.0. N0, initial population. N, population after exposure to acid shock at pH 4.0. N = 15. Bars represent sd. Reproduced from Thomassin et al. (2006), with permission.

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their pHi and DpH (pHi pHo) homeostasis. These ATR mechanisms may involve (i) F0F1-ATPase, glutamate or arginine decarboxylases, arginine deiminase implicated in pHi homeostasis (Jobin, unpublished data), (ii) metabolic modifications (Mols & Abee, 2008) and (iii) synthesis of stress protein to protect, repair or degrade macromolecules. Such mechanisms result in the removal of protons (H+), alkanization of the external environment, changes in the composition of the cell envelope, production of general shock proteins and chaperones, expression of transcriptional regulators, and responses to changes in cell density. These mechanisms counter the negative impact of a reduction in pHi and can all contribute to survival (Cotter & Hill, 2003). ATR can be induced by stress other than acid stress. Cross adaptation of B. cereus was shown as an increased resistance to an acid stress but also to a non-lethal heat-treatment or to inducing concentrations of ethanol, sodium chloride or hydrogen peroxide. Cells were found to maintain pHi at a higher level than the external acid pH and adapted cells had a higher pHi than unadapted cells (Browne & Dowds, 2002).

6. Discussion and conclusion Adaptation mechanisms of B. cereus are very diverse and contribute to its survival (as vegetative cells in a short term or as spores in a longer term) in the environment or to its ability to colonize new ecological niches. Then, the first question is to which extent climate changes (and specifically global warming) could influence the distribution of the genetic groups in the environmental population of B. cereus. von Stetten, Mayr, and Scherer (1999) observed that the prevalence of psychrotrophic strains in soil was correlated with climate, low in tropical soil, intermediate in temperate soil and high in mountain and polar soil. The genetic structure of B. cereus had not been unraveled when this work was published, but it is very likely that it indicates changes in the distribution of the genetic groups in soil according to the climate. Guinebretiere et al. (2008) found that strains, mostly coming from mid and northern Europe, from psychrotrophic groups (genetic groups II and VI) (Table 1) were principally isolated from the environment. This is consistent with von Stetten et al. (1999) finding of the strong association of psychrotrophic B. cereus with soil of temperate and cold areas. However, von Stetten et al. (1999) compared soils from drastically different climates and we have no indication whether subtle changes in climate can cause such a strong and/or rapid impact on psychrotrophic strains. Assuming that climate change could modify the structure of the B. cereus population in soil, the second question is to which extent this will modify the distribution of the various groups at consumer level. Food processing conditions presumably select some genetic groups: according to Guinebretiere et al. (2008); each genetic group of B. cereus tends to be predominantly associated with a ‘‘thermal niche”, the most thermophilic groups with heat processed or dehydrated foods, and psychrotrophic ones primarily with chilled foods. For instance, raw vegetables from Europe carried mostly psychrotrophic groups in contrast to dry ingredients such as starch and dairy proteins (Guinebretiere & Nguyen-The, 2003). In addition to different growth range temperatures, genetic groups also produce spores with different heat resistance (Carlin et al., 2006), spores from the most psychrotrophic groups being the most heat sensitive. Afchain, Carlin, Nguyen-The, and Albert (2008) modeled how a specific food process modifies the structure of B. cereus population from raw foods, containing mostly psychrotrophic groups, to the end product at consumption, containing mostly mesophilic groups. Another example was provided by Svensson et al. (2004) who found that psychrotrophic B. cereus prevailed among isolates from refrigerated pasteurized milk in

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Sweden, in contrast to isolates from dairy farms which were mostly mesophilic. In addition, global food trade presumably makes the B. cereus population in foods less dependant from the local environment. For instance, dry ingredients can be an important source of B. cereus in processed foods (Guinebretiere, Girardin, Dargaignaratz, Carlin, & Nguyen-The, 2003), and such ingredients can be imported from remote countries, e.g. starch from cassava or spices. An impact of global warming on the population of B. cereus in the environment is conceivable as for instance some weather-correlated fluctuations in B. cereus populations have been detected in previous works (Svensson et al., 2004). Which approach would be needed to detect and confirm such changes? Surveys of B. cereus population in foods and food production are regularly published. However, at this step, possible bias related to human activities would need to be considered. In the work from Svensson et al. (2004), authors attributed B. cereus fluctuations to farming practices, i.e. to the seasonal alternation between summer grazing periods (and particularly to a larger exposure of teats to psychrotrophic B. cereus from soil) and winter stall bedding, and not to an effect of the seasonal weather on B. cereus populations in the environment. Moreover an increased detection of B. cereus on vegetable crops may be caused by spreading of the bioinsecticide B. thuringiensis strains (Frederiksen, Rosenquist, Jorgensen, & Wilcks, 2006). Could epidemiological surveillance of B. cereus foodborne poisonings (and of other illnesses) detect an eventual emergence of B. cereus caused by climate change? B. cereus foodborne poisonings are largely underreported and therefore epidemiological surveillance might not be very sensitive to detect changes in B. cereus population. In addition foodborne poisoning depends on what is in food and not directly of what is in the environment. Foodborne illnesses also presumably select ‘‘virulent” B. cereus and might not detect changes in the global population of the bacterium. For instance, if global warming reduces prevalence of B. weihenstephanensis and as it has never been associated so far with foodborne illness, epidemiological surveillance will not detect this change. Therefore surveys considering B. cereus in the environment (e.g. soil and wild life) would be necessary to detect any possible impact of global warming. Such surveys would require an accurate and standardized characterization of B. cereus isolates (for their thermal profile and genetic groups for instance) in long-term followed representative sampling locations. In conclusion, B. cereus represents a unique case of foodborne pathogen with a genetic structure corresponding to groups with different adaptation to temperature. B. cereus is therefore a model of choice to investigate the impact of global warming on bacterial populations. However the experimental sites and the monitoring protocols necessary to detect any possible changes in B. cereus population are presumably not yet in place.

Acknowledgements Some of the original research works exposed in this paper has received support from the Commission of the European Communities, from Agence Nationale de Recherche (France), or from Programme inter-gouvernemental franco-algérien. The authors wish to express their gratitude to their fellow scientists and students who contributed to research works exposed in this presentation.

References Afchain, A. L., Carlin, F., Nguyen-The, C., & Albert, I. (2008). Improving quantitative exposure assessment by considering genetic diversity of B. cereus in cooked, pasteurised and chilled foods. International Journal of Food Microbiology, 128, 165–173.

Aguilar, P. S., Hernandez-Arriaga, A. M., Cybulski, L. E., Erazo, A. C., & de Mendoza, D. (2001). Molecular basis of thermosensing: A two-component signal transduction thermometer in Bacillus subtilis. EMBO Journal, 20, 1681–1691. Alm, E., Huang, K., & Arkin, A. (2006). The evolution of two-component systems in bacteria reveals different strategies for niche adaptation. PLoS Computational Biology, 2, e143. Anderson, I., Sorokin, A., Kapatral, V., Reznik, G., Bhattacharya, A., Mikhailova, N., et al. (2005). Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis. FEMS Microbiology Letters, 250, 175–184. Anonymous (2005). Opinion of the scientific panel on biological hazards on Bacillus cereus and other Bacillus spp. in foodstuffs. The EFSA Journal, 175, 1–48. Auger, S., Galleron, N., Bidnenko, E., Ehrlich, S. D., Lapidus, A., & Sorokin, A. (2008). The genetically remote pathogenic strain NVH391-98 of the Bacillus cereus group is representative of a cluster of thermophilic strains. Applied and Environmental Microbiology, 74, 1276–1280. Baruah, A., Lindsey, B., Zhu, Y., & Nakano, M. M. (2004). Mutational analysis of the signal-sensing domain of ResE histidine kinase from Bacillus subtilis. Journal of Bacteriology, 186, 1694–1704. Baweja, R. B., Zaman, M. S., Mattoo, A. R., Sharma, K., Tripathi, V., Aggarwal, A., et al. (2008). Properties of Bacillus anthracis spores prepared under various environmental conditions. Archives of Microbiology, 189, 71–79. Becker, L. A., Evans, S. N., Hutkins, R. W., & Benson, A. K. (2000). Role of sigma(B) in adaptation of Listeria monocytogenes to growth at low temperature. Journal of Bacteriology, 182, 7078–7083. Brigulla, M., Hoffmann, T., Krisp, A., Volker, A., Bremer, E., & Volker, U. (2003). Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation. Journal of Bacteriology, 185, 4305–4314. Brillard, J., Susanna, K., Michaud, C., Dargaignaratz, C., Gohar, M., Nielsen-Leroux, C., et al. (2008). The YvfTU two-component system is involved in plcR expression in Bacillus cereus. BMC Microbiology, 8. doi:10.1186/1471-2180-1188-1183. Browne, N., & Dowds, B. C. A. (2002). Acid stress in the food pathogen Bacillus cereus. Journal of Applied Microbiology, 92, 404–414. Budde, I., Steil, L., Scharf, C., Volker, U., & Bremer, E. (2006). Adaptation of Bacillus subtilis to growth at low temperature: A combined transcriptomic and proteomic appraisal. Microbiology, 152, 831–853. Carlin, F., Fricker, M., Pielaat, A., Heisterkamp, S., Shaheen, R., Salkinoja Salonen, M., et al. (2006). Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group. International Journal of Food Microbiology, 109, 132–138. Chaturongakul, S., Raengpradub, S., Wiedmann, M., & Boor, K. J. (2008). Modulation of stress and virulence in Listeria monocytogenes. Trends in Microbiology, 16, 388–396. Choma, C., Clavel, T., Dominguez, H., Razafindramboa, N., Soumille, H., Nguyenthe, C., et al. (2000). Effect of temperature on growth characteristics of Bacillus cereus TZ415. International Journal of Food Microbiology, 55, 73–77. Chung, B. H., Cannon, R. Y., & Smith, R. C. (1976). Influence of growth temperature on glucose metabolism of a psychotrophic strain of Bacillus cereus. Applied and Environmental Microbiology, 31, 39–45. Cotter, P. D., & Hill, C. (2003). Surviving the acid test: Responses of Gram-positive bacteria to low pH. Microbiology and Molecular Biology Reviews, 67, 429–453. Davis, M. J., Coote, P. J., & O’Byrne, C. P. (1996). Acid tolerance in Listeria monocytogenes: The adaptive acid tolerance response (ATR) and growthphase-dependent acid resistance. Microbiology, 142, 2975–2982. de Been, M., Bart, M. J., Abee, T., Siezen, R. J., & Francke, C. (2008). The identification of response regulator-specific binding sites reveals new roles of twocomponent systems in Bacillus cereus and closely related low-GC Grampositives. Environmental Microbiology, 10, 2796–2809. de Been, M., Francke, C., Moezelaar, R., Abee, T., & Siezen, R. J. (2006). Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis. Microbiology, 152, 3035–3048. de Vries, Y. P., Atmadja, R. D., Hornstra, L. M., de Vos, W. M., & Abee, T. (2005). Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium. Applied and Environmental Microbiology, 71, 3248–3254. de Vries, Y. P., Hornstra, L. M., Atmadja, R. D., van Schaik, W., de Vos, W. M., & Abee, T. (2005). Deletion of sigB in Bacillus cereus affects spore properties. FEMS Microbiology Letters, 252, 169–173. den Besten, H. M. W., Mols, M., Moezelaar, R., Zwietering, M., & Abee, T. (2009). Phenotype and transcriptome analyses of mildly and severely salt-stressed Bacillus cereus ATCC14579. Applied and Environmental Microbiology, 75, 4111–4119. Dierick, K., Van Coillie, E., Swiecicka, I., Meyfroidt, G., Devlieger, H., Meulemans, A., et al. (2005). Fatal family outbreak of Bacillus cereus-associated food poisoning. Journal of Clinical Microbiology, 43, 4277–4279. Duport, C., Thomassin, S., Bourel, G., & Schmitt, P. (2004). Anaerobiosis and low specific growth rates enhance hemolysin BL production by Bacillus cereus F4430/73. Archives of Microbiology, 182, 90–95. Duport, C., Zigha, A., Rosenfeld, E., & Schmitt, P. (2006). Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system. Journal of Bacteriology, 188, 6640–6651. Eggleton, P., Inward, K., Smith, J., Jones, D. T., & Sherlock, E. (2009). A six year study of earthworm (Lumbricidae) populations in pasture woodland in southern England shows their responses to soil temperature and soil moisture. Soil Biology and Biochemistry, 41, 1857–1865. Ehling-Schulz, M., Svensson, B., Guinebretiere, M.-H., Lindbäck, T., Andersson, M., Schulz, A., et al. (2005). Emetic toxin formation of Bacillus cereus is restricted to

F. Carlin et al. / Food Research International 43 (2010) 1885–1894 a single evolutionary lineage of closely related strains. Microbiology, 151, 183–197. Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer, E., et al. (2005). Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Applied and Environmental Microbiology, 71, 105–113. Esbelin, J., Armengaud, J., Zigha, A., & Duport, C. (2009). ResDE-dependent regulation of enterotoxin gene expression in Bacillus cereus: Evidences for multiple modes of binding for ResD and interaction with Fnr. Journal of Bacteriology, 191, 4419–4426. Esbelin, J., Jouanneau, Y., Armengaud, J., & Duport, C. (2008). ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus. Journal of Bacteriology, 190, 4242–4251. Fagerlund, A., Brillard, J., Furst, R., Guinebretiere, M. H., & Granum, P. E. (2007). Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus group. BMC Microbiology, 7, 43. doi:10.1186/1471-2180-11871143. Faille, C., Tauveron, G., Gentil-Lelievre, C. L., & Slomianny, C. (2007). Occurrence of Bacillus cereus spores with a damaged exosporium: Consequences on the spore adhesion on surfaces of food processing lines. Journal of Food Protection, 70, 2346–2353. Fouet, A., Namy, O., & Lambert, G. (2000). Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence. Journal of Bacteriology, 182, 5036–5045. Francis, K. P., Mayr, R., von Stetten, F., Stewart, G. S. A. B., & Scherer, S. (1998). Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes. Applied and Environmental Microbiology, 64, 3525–3529. Frederiksen, K., Rosenquist, H., Jorgensen, K., & Wilcks, A. (2006). Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensisbased insecticides on fresh fruits and vegetables. Applied Environmental Microbiology, 72, 3435–3440. Gale, P., Drew, T., Phipps, L. P., David, G., & Wooldridge, M. (2009). The effect of climate change on the occurrence and prevalence of livestock diseases in Great Britain: A review. Journal of Applied Microbiology, 106, 1409–1423. Galperin, M. Y. (2005). A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts. BMC Microbiology, 5. doi:10.1186/1471-2180-1185-1135. Gonzalez, I., Lopez, M., Martinez, S., Bernardo, A., & Gonzalez, J. (1999). Thermal inactivation of Bacillus cereus spores formed at different temperatures. International Journal of Food Microbiology, 51, 81–84. Gounina-Allouane, R., Broussolle, V., & Carlin, F. (2008). Influence of the sporulation temperature on the impact of the nutrients inosine and L-alanine on Bacillus cereus spore germination. Food Microbiology, 25, 202–206. Granum, P. E. (2007). Bacillus cereus. In M. P. Doyle & L. R. Beuchat (Eds.), Food microbiology fundamentals and frontiers (3rd ed., pp. 445–455). Washington, DC: ASM Press. Guinebretiere, M. H., Broussolle, V., & Nguyen-The, C. (2002). Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical Microbiology, 40, 3053–3056. Guinebretiere, M. H., Girardin, H., Dargaignaratz, C., Carlin, F., & Nguyen-The, C. (2003). Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked, pasteurized and chilled zucchini puree processing line. International Journal of Food Microbiology, 82, 223–232. Guinebretiere, M. H., & Nguyen-The, C. (2003). Sources of Bacillus cereus contamination in a pasteurized zucchini puree processing line, differentiated by two PCR-based methods. FEMS Microbiology Ecology, 43, 207–215. Guinebretiere, M. H., Thompson, F. L., Sorokin, A., Normand, P., Dawyndt, P., EhlingSchulz, M., et al. (2008). Ecological diversification in the Bacillus cereus group. Environmental Microbiology, 10, 851–865. Haque, M. A., & Russell, N. J. (2004). Strains of Bacillus cereus vary in the phenotypic adaptation of their membrane lipid composition in response to low water activity, reduced temperature and growth in rice starch. Microbiology, 150, 1397–1404. Helgason, E., Tourasse, N. J., Meisal, R., Caugant, D. A., & Kolsto, A. B. (2004). Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Applied and Environmental Microbiology, 70, 191–201. Helmann, J. D. (2002). The extracytoplasmic function (ECF) sigma factors. Advances in Microbial Physiology, 46, 47–110. Hendriksen, N. B., & Hansen, B. M. (2002). Long-term survival and germination of Bacillus thuringiensis var. kurstaki in a field trial. Canadian Journal of Microbiology, 48, 256–261. Hoch, J. A. (2000). Two-component and phosphorelay signal transduction. Current Opinion in Microbiology, 3, 165–170. Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., et al. (2003). Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature, 423, 87–91. Jacobson, M. Z. (2005). Studying ocean acidification with conservative, stable numerical schemes for nonequilibrium air–ocean exchange and ocean equilibrium chemistry. Journal of Geophysical Research, 110. doi:10.1029/ 2004JD005220. Jensen, G. B., Hansen, B. M., Eilenberg, J., & Mahillon, J. (2003). The hidden lifestyles of Bacillus cereus and relatives. Environmental Microbiology, 5, 631–640. Jobin, M. P., Clavel, T., Carlin, F., & Schmitt, P. (2002). Acid tolerance response is lowpH and late-stationary growth phase inducible in Bacillus cereus TZ415. International Journal of Food Microbiology, 79, 65–73.

1893

Konig, H. (2006). Bacillus species in the intestine of termites and other soil invertebrates. Journal of Applied Microbiology, 101, 620–627. Kramer, J. M., & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In M. P. Doyle (Ed.), Foodborne bacterial pathogens (pp. 21–70). New York: Marcel Dekker. Lapidus, A., Goltsman, E., Auger, S., Galleron, N., Segurens, B., Dossat, C., et al. (2008). Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity. Chemico-Biological Interactions, 171, 236–249. Lechner, S., Mayr, R., Francis, K. P., Prüss, B., Kaplan, T., Wiessner-Gunkel, E., et al. (1998). Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. International Journal of Systematic Bacteriology, 48, 1373–1382. Leguerinel, I., Couvert, O., & Mafart, P. (2007). Modelling the influence of the sporulation temperature upon the bacterial spore heat resistance, application to heating process calculation. International Journal of Food Microbiology, 114, 100–104. Lund, T., De Buyser, M. L., & Granum, P. E. (2000). A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology, 38, 254–261. Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., Baer, W., et al. (1997). Fulminant liver failure in association with the emetic toxin of Bacillus cereus. The New England Journal of Medicine, 336, 1142–1148. Margulis, L., Jorgensen, J. Z., Dolan, S., Kolchinsky, R., Rainey, F. A., & Lo, S. C. (1998). The Arthromitus stage of Bacillus cereus: Intestinal symbionts of animals. Proceedings of the National Academy of Sciences of the United States of America, 95, 1236–1241. Marles-Wright, J., & Lewis, R. J. (2007). Stress responses of bacteria. Current Opinion in Structural Biology, 17, 755–760. Mayr, B., Kaplan, T., Lechner, S., & Scherer, S. (1996). Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201. Journal of Bacteriology, 178, 2916–2925. Mazas, M., Fernandez, A., Alvarez, A., Lopez, M., & Bernardo, A. (2009). Effects of phosphate and sodium and potassium chlorides on sporulation and heat resistance of Bacillus cereus. Journal of Food Safety, 29, 106–117. McLaughlin, J. B., DePaola, A., Bopp, C. A., Martinek, K. A., Napolilli, N. P., Allison, C. G., et al. (2005). Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. New England Journal of Medicine, 353, 1463–1470. McMichael, A. J., Woodruff, R. E., & Hales, S. (2006). Climate change and human health: Present and future risks. Lancet, 367, 859–869. Mols, M., & Abee, T. (2008). Role of ureolytic activity in Bacillus cereus nitrogen metabolism and acid survival. Applied and Environmental Microbiology, 74, 2370–2378. Mols, M., de Been, M., Zwietering, M. H., Moezelaar, R., & Abee, T. (2007). Metabolic capacity of Bacillus cereus strains ATCC 14579 and ATCC 10987 interlinked with comparative genomics. Environmental Microbiology, 9, 2933–2944. Moravek, M., Dietrich, R., Buerk, C., Broussolle, V., Guinebretiere, M. H., Granum, P. E., et al. (2006). Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses. FEMS Microbiology Letters, 257, 293–298. Moriarty-Craige, S. E., & Jones, D. P. (2004). Extracellular thiols and thiol/disulfide redox in metabolism. Annual Review of Nutrition, 24, 481–509. Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J., & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and Molecular Biology Reviews, 64, 548–572. Nishi, J. S., Dragon, D. C., Elkin, B. T., Mitchell, J., Ellsworth, T. R., & Hugh-Jones, M. E. (2002). Emergency response planning for anthrax outbreaks in bison herds of northern Canada – A balance between policy and science. In Domestic animal/ wildlife interface. Issue for disease control, conservation, sustainable food production, and emerging diseases conference on wildlife and livestock, disease and sustainability, July 22–27, 2001 Pilanesberg Natl Pk, South Africa (pp. 245–250). New York: New York Academy of Sciences. Normand, P., Lapierre, P., Tisa, L. S., Gogarten, J. P., Alloisio, N., Bagnarol, E., et al. (2007). Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography. Genome Research, 17, 7–15. Ohara, G. W., & Glenn, A. R. (1994). The adaptive acid tolerance response in rootnodule bacteria and Escherichia coli. Archives of Microbiology, 161, 286–292. Ouhib, O., Clavel, T., & Schmitt, P. (2006). The production of Bacillus cereus enterotoxins is influenced by carbohydrate and growth rate. Current Microbiology, 53, 222–226. Ouhib-Jacobs, O., Lindley, N. D., Schmitt, P., & Clavel, T. (2009). Fructose and glucose mediates enterotoxin production and anaerobic metabolism of Bacillus cereus ATCC14579T. Journal of Applied Microbiology, 107, 821–829. Periago, P. M., Abee, T., & Wouters, J. A. (2002). Analysis of the heat-adaptive response of psychrotrophic Bacillus weihenstephanensis. International Journal of Food Microbiology, 79, 17–26. Periago, P., van Schaik, W., Abee, T., & Wouters, J. A. (2002). Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579. Applied and Environmental Microbiology, 68, 3486–3495. Piggot, P. J., & Hilbert, D. W. (2004). Sporulation of Bacillus subtilis. Current Opinion in Microbiology, 7, 579–586. Priest, F. G., Barker, M., Baillie, L. W. J., Holmes, E. C., & Maiden, M. C. J. (2004). Population structure and evolution of the Bacillus cereus group. Journal of Bacteriology, 186, 7959–7970. Pruss, B. M., Francis, K. P., von Stetten, F., & Scherer, S. (1999). Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group. Journal of Bacteriology, 181, 2624–2630.

1894

F. Carlin et al. / Food Research International 43 (2010) 1885–1894

Raso, J., Gongora-Nieto, M. M., Barbosa Canovas, G. V., & Swanson, B. G. (1998). Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure. International Journal of Food Microbiology, 44, 125–132. Rau, J., Perz, R., Klittich, G., & Contzen, M. (2009). Cereulide forming presumptive Bacillus cereus strains from food – Differentiating analyses using cultural methods, LC–MS/MS, PCR, and infrared spectroscopy in consideration of thermotolerant isolates. Berliner Und Munchener Tierarztliche Wochenschrift, 122, 25–36. Raymond, B., Lijek, R. S., Griffiths, R. I., & Bonsall, M. B. (2008). Ecological consequences of ingestion of Bacillus cereus on Bacillus thuringiensis infections and on the gut flora of a lepidopteran host. Journal of Invertebrate Pathology, 99, 103–111. Reents, H., Gruner, I., Harmening, U., Bottger, L. H., Layer, G., & Heathcote, P. (2006). Bacillus subtilis Fnr senses oxygen via a [4Fe–4S] cluster coordinated by three cysteine residues without change in the oligomeric state. Molecular Microbiology, 60, 1432–1445. Root, T. L., Price, J. T., Hall, K. R., Schneider, S. H., Rosenzweig, C., & Pounds, J. A. (2003). Fingerprints of global warming on wild animals and plants. Nature, 421, 57–60. Rosenfeld, E., Duport, C., Zigha, A., & Schmitt, P. (2005). Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain. Canadian Journal of Microbiology, 51, 149–158. Rosenzweig, C., Karoly, D., Vicarelli, M., Neofotis, P., Wu, Q., Casassa, G., et al. (2008). Attributing physical and biological impacts to anthropogenic climate change. Nature, 453, 353–357. Schoeni, J. L., & Wong, A. C. L. (2005). Bacillus cereus food poisoning and its toxins. Journal of Food Protection, 68, 636–648. Schumann, W. (2009). Temperature sensors of Eubacteria. Advances in Applied Microbiology, 67, 213–256. Setlow, P., & Johnson, E. A. (2007). Spores and their significance. In M. P. Doyle & L. R. Beuchat (Eds.), Food microbiology fundamentals and frontiers (pp. 35–68). Washington, DC: ASM Press. Sorokin, A., Candelon, B., Guilloux, K., Galleron, N., Wackerow-Kouzova, N., Ehrlich, S. D., et al. (2006). Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Applied and Environmental Microbiology, 72, 1569–1578. Stecchini, M. L., Spaziani, M., Del Torre, M., & Pacor, S. (2009). Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium. Journal of Applied Microbiology., 106, 1838–1848. Stephenson, K., & Hoch, J. A. (2004). Developing inhibitors to selectively target twocomponent and phosphorelay signal transduction systems of pathogenic microorganisms. Current Medicinal Chemistry, 11, 765–773. Svensson, B., Ekelund, K., Ogura, H., & Christiansson, A. (2004). Characterisation of Bacillus cereus isolated from milk silo tanks at eight different dairy plants. International Dairy Journal, 14, 17–27. Svensson, B., Monthan, A., Guinebretiere, M. H., Nguyen-The, C., & Christiansson, A. (2007). Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain. International Dairy Journal, 17, 1201–1208. Swiecicka, I. (2008). Natural occurrence of Bacillus thuringiensis and Bacillus cereus in eukaryotic organisms: A case for symbiosis. Biocontrol Science and Technology, 18, 221–239. Taylor, B. L., & Zhulin, I. B. (1999). PAS domains: Internal sensors of oxygen, redox potential, and light. Microbiology and Molecular Biology Reviews, 63, 479–506.

Te Giffel, M. C., Beumer, R. R., Granum, P. E., & Rombouts, F. M. (1997). Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in the Netherlands. International Journal of Food Microbiology, 34, 307–318. Thomassin, S., Jobin, M. P., & Schmitt, P. (2006). The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH. Archives of Microbiology, 186, 229–239. Tiwari, R. P., Sachdeva, N., Hoondal, G. S., & Grewal, J. S. (2004). Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi. Journal of Basic Microbiology, 44, 137–146. Trenberth, K. E., Jones, P. D., Ambenje, P., Bojariu, R., Easterling, D., Klein Tank, A., et al. (2007). Observations: Surface and atmospheric climate change. In S. Solomon, D. Qin, M. Manning, Z. Chen, M. Marquis, & K. B. Averyt, et al. (Eds.), Climate change 2007: The physical science basis contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change. Cambridge, United Kingdom and New York, NY, USA: Cambridge University Press. van Schaik, W., & Abee, T. (2005). The role of sigma(B) in the stress response of Gram-positive bacteria – Targets for food preservation and safety. Current Opinion in Biotechnology, 16, 218–224. van Schaik, W., Tempelaars, M. H., Wouters, J. A., de Vos, W. M., & Abee, T. (2004). The alternative sigma factor sigmaB of Bacillus cereus: Response to stress and role in heat adaptation. Journal of Bacteriology, 186, 316–325. van Schaik, W., van der Voort, M., Molenaar, D., Moezelaar, R., de Vos, W. M., & Abee, T. (2007). Identification of the sigma(B) regulon of Bacillus cereus and conservation of sigma(B)-regulated genes in low-GC-content Gram-positive bacteria. Journal of Bacteriology, 189, 4384–4390. van Schaik, W., Zwietering, M. H., de Vos, W. A., & Abee, T. (2005). Deletion of the sigB gene in Bacillus cereus ATCC 14579 leads to hydrogen peroxide hyperresistance. Applied and Environmental Microbiology, 71, 6427–6430. Vassileva, M., Torii, K., Oshimoto, M., Okamoto, A., Agata, N., Yamada, K., et al. (2007). A new phylogenetic cluster of cereulide-producing Bacillus cereus strains. Journal of Clinical Microbiology, 45, 1274–1277. Vilain, S., Luo, Y., Hildreth, M. B., & Brozel, V. S. (2006). Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Applied and Environmental Microbiology, 72, 4970–4977. von Stetten, F., Mayr, R., & Scherer, S. (1999). Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil. Environmental Microbiology, 1, 503–515. Wiegeshoff, F., Beckering, C. L., Debarbouille, M., & Marahiel, M. A. (2006). Sigma L is important for cold shock adaptation of Bacillus subtilis. Journal of Bacteriology, 188, 3130–3133. Wilcks, A., Smidt, L., Bahl, M. I., Hansen, B. M., Andrup, L., Hendriksen, B., et al. (2008). Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats. Journal of Applied Microbiology, 104, 1252–1259. Zaller, J. G., Caldwell, M. M., Flint, S. D., Ballare, C. L., Scopel, A. L., & Sala, O. E. (2009). Solar UVB and warming affect decomposition and earthworms in a fen ecosystem in Tierra del Fuego, Argentina. Global Change Biology, 15, 2493–2502. Zigha, A., Rosenfeld, E., Schmitt, P., & Duport, C. (2006). Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression. Archives of Microbiology, 185, 222–233. Zigha, A., Rosenfeld, E., Schmitt, P., & Duport, C. (2007). The redox regulator Fnr is required for fermentative growth and enterotoxin synthesis in Bacillus cereus F4430/73. Journal of Bacteriology, 189, 2813–2824.