Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms

Sporulation environment influences spore properties in Bacillus: evidence and insights on... Abstract Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health and industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties. resistance, germination, sporulation environment, structure, exosporium, coat INTRODUCTION Spores are forms of resistance of Bacillus sp. and other Firmicutes. The elimination of bacterial spores of pathogenic species in healthcare facilities remains a problematic issue that hampers efforts to prevent nosocomial infections (Bottone 2010; Maillard 2011; Barra-Carrasco and Paredes-Sabja 2014). The thermal intensity of food-processing operations (in the canning industry or in ultra-high-temperature processing) is also designed to efficiently inactivate bacterial spores. Even so, spores may still survive, which means many unprocessed or processed foods depend on in-storage refrigeration for bacterial safety or prevention of spoilage (Logan 2012; Wells-Bennik et al.2016). That said, spore-forming bacteria also have many desirable properties (Wolken, Tramper and van der Werf 2003). Their vegetative and/or sporulating cells produce enzymes and metabolites that are exploited for applications in agronomics, veterinary medicine, healthcare and biotech. For example Bacillusthuringiensis is in widespread use for the bioinsecticidal properties of toxins it forms during sporulation and Bacillus sp. are also exploited as plant-growth promoters or bio-fertilizers (Perez-Garcia, Romero and de Vicente 2011). At industrial scale, spore resistance favors the formulation and storage of commercial products at ambient temperature for extended periods with only limited effects on spore viability. Regarding animal health, Bacillus probiotics are used in humans as dietary supplements and in livestock, poultry or aquaculture as growth or disease resistance promoters and competitive exclusion agents (Cutting 2011). Probiotic spores are also attractive delivery vehicle for oral vaccination thanks to resistance to gastric acidity (Rosales-Mendoza, Angulo and Meza 2016). Bacterial spores that borrow genomic roots from the Firmicutes phylum are the most resistant form of life on Earth. Spores can remain dormant and metabolically inert for very long periods and longevity and lifetimes of hundreds to thousands year have been reported in several instances (Nicholson 2003; Gould 2006). Spores can be dispersed by wind, water, or living hosts, and by transportation of natural or industrial material to locations far away from the sporulation site, and ultimately in an environment not immediately suitable for growth. For example, the presence of thermophilic spores in cold environments can be attributed to dispersion from geothermal aquifers and hydrothermal vents (de Rezende et al.2013). When the environment becomes favorable, spores can break dormancy and re-initiate a lifecycle through germination and outgrowth processes. This lifecycle represents a successful mechanism for widespread dispersal of spore-forming bacteria on Earth. Thus spores are found in highly diverse environmental niches, from abiotic and biotic fractions of soil including the rhizosphere to the gut of terrestrial and aquatic animals including mammals and on to industrial installations and healthcare facilities. They thus are exposed to a huge diversity of adverse conditions, from extreme temperature swings, eventually freezing and thawing, to physical abrasion, desiccation or exposure to corrosive chemicals, solar or industrial radiation, or even predation (Nicholson et al.2000). The sporulation process generates a spore that has a radically different structure to the vegetative cell (Fig. 1). The outermost ‘balloon-like’ layer, called exosporium, is found in some Bacillus species, such as Bacillus cereus or Bacillus anthracis. This first point of spore contact with the environment is highly hydrophobic and allows the spores to adhere to cells and abiotic surfaces (Oliva, Turnbough and Kearney 2009; Lequette et al.2011; Xue et al.2011; Stewart 2015). Bacillus subtilis has no exosporium—its outermost structure is a proteic ‘crust’ (Henriques and Moran 2007; Imamura et al.2010; McKenney et al.2010; Leggett et al.2012; McKenney, Driks and Eichenberger 2013). The B. subtilis spore coat is a protein-rich structure composed of an inner and outer layer separated in species possessing an exosporium by a large ‘interspace’ (Giorno et al.2007; Leggett et al.2012; Setlow 2014a,b). The relative permeability, to lysozyme for instance, of the outer membrane beneath the spore coat does not likely confer resistance to spores (Setlow 2006). The peptidoglycan of the spore cortex is required for the maintenance of spore core dehydration and also plays a role in dormancy (Foster and Popham 2002). The inner membrane that protects the spore core is characterized by low permeability to small molecules and water (Paredes-Sabja and Sarker 2011; Bassi, Cappa and Cocconcelli 2012). The spore core contains DNA encased in small acid-soluble spore proteins (SASP), high levels of dipicolinic acid (DPA) chelated to divalent Ca2+ cations (CaDPA), and minerals, and is characterized by a low water content (Setlow 2014a,b). The assembly and final arrangement of these different spore structures confers survival and persistence through resistance and dormancy, pending suitable conditions for growth. Figure 1. View largeDownload slide Structure of a Bacillus cereus spore and roles of the spore components in resistance. (A) Negative staining image, (B) transmission electron microscopy image of thin section and (C) schematic view of a B. cereus spore structure. The exosporium (Ex) is a balloon-like structure loosely anchored to the coat through protein-protein interactions. The exosporium contributes to spore attachment to abiotic and biotic surfaces. Spore appendages (Sa) are clearly visible on panel (A). The coat (Ct) is separated from the exosporium by an interspace (Is). The coat represents a large part of total spore proteins, organized as a permeability barrier to degradative enzymes, and detoxifies deleterious chemicals. The coat protects the innermost spore components and maintains low water permeability. The outer membrane (Om) may accumulate carotenoids, pigments protecting the spore against UV radiations. The cortex (Cx) is made of peptidoglycan. Its role in resistance is unknown, but it may be implicated in the low water content of the spore core. The low permability of the inner membrane (Im) contributes to the protection against disinfectants and some DNA damaging chemicals. The core (Co) contains the DNA saturated with α/β type SASP protecting against UV- and γ-radiation, dry heat and wet heat, genotoxic chemicals and some oxidizing agents. Spore core has a low water content, a high level of DPA and divalent metals protecting against desiccation, dry and moist heat. Photographic images are from INRA Avignon, France. Adapted from Driks (1999), Nicholson et al. (2000), Melly et al. (2002), Setlow et al. (2006), Setlow (2014b), Stewart (2015) and Knudsen et al. (2016). Figure 1. View largeDownload slide Structure of a Bacillus cereus spore and roles of the spore components in resistance. (A) Negative staining image, (B) transmission electron microscopy image of thin section and (C) schematic view of a B. cereus spore structure. The exosporium (Ex) is a balloon-like structure loosely anchored to the coat through protein-protein interactions. The exosporium contributes to spore attachment to abiotic and biotic surfaces. Spore appendages (Sa) are clearly visible on panel (A). The coat (Ct) is separated from the exosporium by an interspace (Is). The coat represents a large part of total spore proteins, organized as a permeability barrier to degradative enzymes, and detoxifies deleterious chemicals. The coat protects the innermost spore components and maintains low water permeability. The outer membrane (Om) may accumulate carotenoids, pigments protecting the spore against UV radiations. The cortex (Cx) is made of peptidoglycan. Its role in resistance is unknown, but it may be implicated in the low water content of the spore core. The low permability of the inner membrane (Im) contributes to the protection against disinfectants and some DNA damaging chemicals. The core (Co) contains the DNA saturated with α/β type SASP protecting against UV- and γ-radiation, dry heat and wet heat, genotoxic chemicals and some oxidizing agents. Spore core has a low water content, a high level of DPA and divalent metals protecting against desiccation, dry and moist heat. Photographic images are from INRA Avignon, France. Adapted from Driks (1999), Nicholson et al. (2000), Melly et al. (2002), Setlow et al. (2006), Setlow (2014b), Stewart (2015) and Knudsen et al. (2016). Many spore-forming species sporulate in laboratory conditions. The range of media and incubation environments that support sporulation is huge, but how far does it influence spore properties like resistance? This question addresses fundamental issues of how intensively metabolic, physiological or molecular adaptations during growth will interfere with sporulation and final spore properties. But it also has practical implications: many applications, such as risk assessment in foods, analysis of disinfectant efficiency, probiotics for health, crop pest control, or design of bioindicators, use spores that are usually formed in artificial environments. How reliable are inactivation, survival, germination and growth predictions for food safety or hygiene of healthcare facilities based on cultivated spores compared to spores formed in natural environments? How do sporulation conditions impact the gastric survival of spores administered as probiotics and their interactions with the gut microbiota and mucosa? The aim of this review is to illustrate how sporulation conditions affect the spore properties of Bacillus sp., and to identify, when possible, the most plausible molecular and physiological mechanisms behind the observed effects. Environmental conditions influence sporulation efficiency, spore structure and composition Factors modulating spore formation In response to nutrient limitation and quorum sensing signals, vegetative cells of Bacillus species transform into sporulating cells after an asymmetric division through a complex developmental process. A phosphorelay regulatory system activates the sporulation pathway through transcription factors that are phosphorylated by sensor kinases (Sonenshein 2000; Hilbert and Piggot 2004; Higgins and Dworkin 2012; Tan and Ramamurthi 2014; Decker and Ramamurthi 2017). Sporulation yield can be estimated as the amount of vegetative cells that undergo a complete sporulation process. Spores are enumerated by plate-counting heat-resistant cells in a suspension, or by counting refractile cells with a phase-contrast microscope or a flow cytometer. Unsurprisingly, the environmental factors that influence growth of Bacillus cells also deeply affect sporulation. To the best of our knowledge, there is still no evidence that Bacillus spores can be formed outside the range of temperature, pH and water activity (aw) allowing growth. Sporulation yield is usually maximal at optimal growth temperature, pH or aw, and decreases as temperature, pH or aw stray from optimum, which tends to lengthen the sporulation process (Mazas et al.1997; Baweja et al.2008; Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Garcia, van der Voort and Abee 2010; Nguyen Thi Minh et al.2011; Planchon et al.2011; Baril et al.2012). For instance, the time to complete growth and sporulation of B. subtilis ATCC31324 is 3 days at 37°C, pH 8.0 and high aw in a standard nutrient broth (optimal conditions). It increases to 10 days at 45°C and 14 days at 19°C, to 20 days at pH 6.0 or pH 10.0, and to 17 days at aw = 0.950 (Nguyen Thi Minh et al.2011). Observations on Bacillus weihenstephanensis and Bacillus licheniformis suggested that temperature and pH could have the same quantitative influence on both maximum specific growth rate and sporulation rate (Baril et al.2012). Counts of Bacillus sp. spores were generally maximal at optimal temperature, pH and aw and remained high in a wide range of culture conditions (Mazas et al.1997; Baweja et al.2008; Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Garcia, van der Voort and Abee 2010; Nguyen Thi Minh et al.2011). Sporulation yield of the psychrotrophic B. weihenstephanensis KBAB4, which has a minimal temperature for growth of approximately 6°C, was >99% at 12°C and at 30°C (Garcia, van der Voort and Abee 2010), whereas spore formation was also observed at 10°C and 7°C but with much lower efficiency. Inhibition of B. subtilis sporulation by high salinity (about 7% NaCl) occurs at an early stage due to impaired activity of the response regulator Spo0A governing entry into sporulation and of the alternate sigma factor σH (Widderich et al.2016). In many instances, total spore count and sporulation yield remain relatively unchanged as conditions approach the limits of sporulation and growth. However, sporulation, which can be high within a large range of conditions allowing growth, varies strongly with strain, sporulation media and incubation conditions. Sporulation yields for B. thuringiensis and B. cereus, for example, are affected by oxygen concentration: the quantity of spores formed (and cell growth) were lower under oxygen limitation than under aerobiosis (Avignone-Rossa, Arcas and Mignone 1992; Boniolo et al.2012; Abbas et al.2014). The sporulation medium's level of nutrients, in particularly mono/di-valent cations, is also a major factor. Supplementation with Mn2+, Mg2+, Zn2+, Ca2+ increased the sporulation yield of Bacillus species and improved the stability of spores which no longer underwent spontaneous germination (Atrih and Foster 2001). Time to complete full sporulation of B. subtilis was almost five times longer in absence than in presence of Ca2+ (Nguyen Thi Minh et al.2011). It has long been known that the sporulation process and final spore yield are amino acid- and carbohydrate-dependent (Schaeffer et al.1965). The combined effects of yeast extract, peptone and glucose enhanced the spore yield of B. megaterium (Verma et al.2013). Likewise, the addition of glucose and ribose in the sporulation medium increased the spore yield of B. subtilis and B. cereus (Warriner and Waites 1999; de Vries et al.2005; Monteiro et al.2005). Optimizing glucose and Mg2+concentration in a sporulation medium led to a 17-fold increase in spore yield of a B. subtilis strain promoting plant growth (Posada-Uribe, Romero-Tabarez and Villegas-Escobar 2015). The mechanism by which cell metabolism affects sporulation is complex. For instance, the sporulation process of B. cereus ATCC14579 was substantially longer in presence of high glutamate concentration (de Vries et al.2005). However, different glutamate concentrations had no effect on the temporal expression of sigF and sigG genes encoding the key transcriptional sporulation factors σF and σG. Glutamate concentration may therefore affect the programming of sporulation events that occur after those directly controlled by σG, which include mother cell lysis and spore maturation. Factors influencing spore structure and composition Spore structure and composition are also affected by changes in the sporulation environment (Fig. 2). Spores accumulate minerals in the spore core (mainly Ca2+, Mg2+ and Mn2+) during the sporulation process. Spore-core mineral concentrations vary widely and are highly dependent on the composition of the sporulation medium (Bassi, Cappa and Cocconcelli 2012). The spore core is also characterized by a high content of CaDPA that may form, according to a recently proposed model, an inorganic polymer bridged by water molecules that maintains the spore core in an as-yet undetermined state described as glassy or gel-like (Setlow and Li 2015). This water-CaDPA polymer favors spore resistance by immobilizing proteins or molecules such as membrane lipids (Cowan et al.2003; Cowan et al.2004). Bacillussubtilis spores lacking the ability to synthesize DPA and that are formed in a growth medium without added DPA have a much higher core water content than spores formed in a DPA-supplemented medium (Paidhungat et al.2000). Spores of several Bacillus have a lower water content when formed on nutrient agar with a mix of metal cations (Ca2+, Mg2+, Fe2+, K+ and Mn2+) than with Mn2+ only or without cations (Cazemier, Wagenaars and ter Steeg 2001; Nguyen Thi Minh et al.2011). Sporulation temperature is also among the factors that have the strongest effect on DPA and core water content, although this effect may not be systematically reported (Melly et al.2002; Kaieda et al.2013). DPA concentrations were higher in B. cereus and B. anthracis spores formed at high temperatures (30°C and 45°C, respectively) rather than at low temperatures (10°C and 25°C, respectively) (Baweja et al.2008; Planchon et al.2011). Water content was lower in B. subtilis spores formed at high temperature than at low temperature (Beaman and Gerhardt 1986; Melly et al.2002; Nguyen Thi Minh et al.2011). Moreover higher sporulation temperature has also been correlated with higher levels of core spore mineralization (Palop, Sala and Condon 1999; Igura et al.2003). The cortex peptidoglycan of spores prepared at different temperatures showed subtle changes in structure, with slightly increased percentages of cross-linked muramic acid in spores prepared at high temperatures (Melly et al.2002). Cortex peptidoglycan structure was also substantially modified in spores of various Bacillus species produced in a nutrient-poor medium without any carbon source compared to a rich medium (Atrih and Foster 2001). The authors hypothesized that a change in muropeptide ratio, as well as a probable decrease in number of muropeptides containing δ-lactam, reveals a functional defect of the cortex biosynthesis pathway and/or maturation machinery. Likewise, addition of MnCl2 to sporulation media resulted in an altered peptidoglycan composition and peptidoglycan chain cross-linking. Mn2+ may therefore affect the expression of genes and/or the activity of enzymes involved in cortex biosynthesis (Atrih and Foster 2001). Figure 2. View largeDownload slide A synthetic view on the major factors known to influence structure and composition of Bacillus spores. indicates an effect on a spore component or structure. Effect on a spore component or structure with consequences on resistance to heat is indicated by , with consequences on resistance to chemical biocides by , and with consequences on spore germination by . Coat P, coat proteins. Ger, germinant receptors. DPA, dipicolinic acid. M+, metal ions. H2O, spore core water content. Ex, exosporium. Is, interspace. Ct, coat. Cx, cortex. Im, Om, inner and outer membrane. Co, core. Figure 2. View largeDownload slide A synthetic view on the major factors known to influence structure and composition of Bacillus spores. indicates an effect on a spore component or structure. Effect on a spore component or structure with consequences on resistance to heat is indicated by , with consequences on resistance to chemical biocides by , and with consequences on spore germination by . Coat P, coat proteins. Ger, germinant receptors. DPA, dipicolinic acid. M+, metal ions. H2O, spore core water content. Ex, exosporium. Is, interspace. Ct, coat. Cx, cortex. Im, Om, inner and outer membrane. Co, core. Variations in sporulation temperature, pH or concentration of inorganic salts resulted in substantial variations in spore volumes, which ranged from 0.38 μm3; to 0.79 μm3; for B. cereus and 0.53 μm3; to 0.71 μm3; for B. megaterium (Zhou et al.2017). Bacillussubtilis spores were nearly twice as small when formed in a Ca2+-deprived sporulation medium (Nguyen Thi Minh et al.2011). Spores of several B. cereus strains produced in liquid media were significantly smaller than the ones formed on agar or in biofilms (van der Voort and Abee 2013). The roughness of the spore surface can be affected by incubation temperature or by aw of the sporulation medium (Nguyen Thi Minh et al.2011). Spore swelling (shrinking) in response to high (low) relative humidity and core (de)hydration of B. thuringiensis and B. subtilis spores could suggest a link between spore (de)hydration and spore size (Westphal et al.2003; Sunde et al.2009). However, spore hydration is not the only cause: varying sporulation conditions led to marked differences in spore volumes but without any difference in spore wet density (Zhou et al.2017). Other structural modifications are directly observable with electron microscopy. Examples are that B. cereus exosporium is damaged and detached when spores are formed at high temperature (Faille et al.2007), or that the outer coat of B. subtilis spores is thicker in spores formed in a chemically-defined broth than in a rich agar medium (Abhyankar et al.2016). Differences in coat protein profiles were observed for B. subtilis spores prepared at various temperatures or in broth vs. agar plates (Melly et al.2002; Rose et al.2007; Abhyankar et al.2016). In contrast α/β-type SASP remained unaffected in the same sporulation conditions (Melly et al.2002; Rose et al.2007). The intensity of the electrophoretic bands of the coat proteins CotA, CotG, CotB and CotS was lower in extracts from spores prepared at 22°C and 30°C than at 48°C. Several proteins of coat and exosporium extracts from B. cereus differed in spores formed at 20°C or 37°C (Bressuire-Isoard et al.2016). Among these, the CotE protein was proportionally greater in extracts from spores produced at 20°C than at 37°C. These differences may result from differences in the amount and/or extractability of the proteins. In B. subtilis for instance, protein extraction yielded a higher amount of total proteins from spores prepared on agar than from spores prepared in broth (Rose et al.2007). Nevertheless, relative coat protein contents did not show any major difference between spores formed in agar vs. in broth (Driks 1999; Rose et al.2007). Bacilluscereus and B. subtilis spores produced at different sporulation temperatures showed significant differences in fatty acid (FA) composition. Total amount of anteiso FA increased in B. cereus spores produced at low temperature (Planchon et al.2011). The anteiso-to-iso ratio and the amount of unsaturated FA in B. subtilis spore inner membrane increased as temperature decreased (Cortezzo and Setlow 2005). These changes correspond to those generally observed in Bacillus spp. cells during low-temperature adaptation (Diomande et al.2015) and likely reflect a need to maintain membrane fluidity. A similar higher anteiso-to-iso FA ratio in the inner membrane was observed for spores formed on plates vs. broth (Rose et al.2007). Spore structure, organization and composition are clearly very sensitive to changes in the physical and chemical sporulation environment. Sporulation conditions affect spore resistance Many spore structures are involved in spore resistance Resistance to extreme temperatures is a distinguishing property between bacterial spores and vegetative cells, as wet-heat inactivation of spores requires a roughly 45°C higher temperature than wet-heat inactivation of vegetative cells (Setlow 2014a,b; Checinska, Paszczynski and Burbank 2015). Spore killing by wet heat is mainly due to damages to core proteins and denaturation of enzymes involved in metabolism (Coleman et al.2007; Setlow 2014a,b; Wells-Bennik et al.2016). Resistance to wet heat involves DNA saturation with α/β-type SASP proteins, low core water, high DPA and mineral content, likely reducing molecular mobility in the core and protecting proteins against thermal denaturation and irreversible aggregation (Sunde et al.2009; Setlow 2014a,b). The spore cortex, through the level of peptidoglycan cross-linking, could also be involved in maintaining the dehydrated state of B. subtilis spore cores (Atrih and Foster 1999; Driks 1999). However, how such modifications in cortex composition actually maintain spore core dehydration remains unknown. Increase in resistance during spore maturation concomitantly with cross-linking of the outer coat proteins has recently suggested a possible role of coat in resistance to wet heat (Sanchez-Salas et al.2011; Abhyankar et al.2015). Spores of at least several Bacillus sp. share different mechanisms for resistance to dry heat and wet heat. Resistance to dry heat is related to DNA saturation by α/β-type SASP in the core and activation of systems to repair dry heat-induced DNA damage during spore outgrowth like the RecA protein (Setlow and Setlow 1996; Nicholson et al.2000; Setlow 2014a,b; Setlow et al.2014). Besides resistance to heat, spores are also resistant to an array of physical insults including desiccation, freeze–thaw cycles, UV and γ-radiation, high hydrostatic pressure and chemical insults involving a variety of toxic effects (Nicholson et al.2000; Setlow 2014a,b; Checinska, Paszczynski and Burbank 2015). Resistance to UV involves two major factors—α/β-type SASP binding to DNA, and DNA repair during spore outgrowth—plus minor factors such as carotenoids in spores outer layers, and low water and high DPA content in spore core (Setlow 2014a,b). Spores are 10–50 times more resistant to UV radiation than vegetative cells, depending on the species studied (Nicholson et al.2000). Strong acid treatments, organic solvents at high temperatures, and oxidizing agents such as hydrogen peroxide all cause major damages in the inner spore membrane, where oxidation of membrane proteins may result in rupture and cell death (Cortezzo et al.2004; Cortezzo and Setlow 2005). Alkali treatments mainly inactivate the lytic enzymes of the coat that hydrolyze the cortex during germination (Setlow et al.2002). The spore coat is the first line of defense against large molecules targeting the spore cortex, and it plays a major role in shielding the spore against oxidizing agents such as chlorine dioxide, hypochlorite or peroxynitrite (Setlow 2000; Genest et al.2002; Melly et al.2002; Young and Setlow 2003). Resistance to chemicals involves a large number of factors, such as detoxifying enzymes of spore coat and/or exosporium including catalase or superoxide dismutase, low permeability of the spore inner membrane, DNA protection by α/β-type SASPs and delete DNA repair systems (Setlow 2006; Setlow 2014a,b). Factors influencing resistance to heat The impact of environmental conditions on spore heat resistance was already observed back in the late 1920s on B. anthracis spores, which were more heat-resistant when formed at 37°C than at 18°C (Williams 1929). Spores of Bacillus sp. prepared at suboptimal temperatures are consistently less resistant to wet heat than spores prepared at near-optimal temperatures (Beaman and Gerhardt 1986; Condon, Bayarte and Sala 1992; Raso et al.1995; González et al.1999; Baweja et al.2008; Garcia, van der Voort and Abee 2010; Baril et al.2011; Nguyen Thi Minh et al.2011; Planchon et al.2011; Bressuire-Isoard et al.2016). The difference can be quite significant. Heat-resistance parameters such as decimal reduction times, or D values, can vary by a factor greater than 10 depending on sporulation temperature (Fig. 3; Table S1, Supporting Information), and some authors have even proposed the concept of an optimal sporulation temperature and pH at which spores get their maximal wet-heat resistance (Baril et al.2012; Trunet et al.2015). Spore core water content decreases as sporulation temperature increases and, as stated earlier under ‘Factors influencing spore structure and composition’, there is a reciprocal positive correlation between core dehydration and spore wet heat resistance. DPA contributes to spore wet heat resistance by replacing water molecules and therefore maintaining low spore core water content. Similarly, a higher sporulation temperature correlated with higher levels of spore mineralization which results in higher heat resistance (Palop, Sala and Condon 1999). Sporulation temperature has subtle effects on the cortex structure of B. subtilis spores, with potential changes in core hydration (Melly et al.2002). Even a few minutes heat-shock or cold-shock at an appropriate time during sporulation can affect wet heat resistance. However, heat-shock and cold-shock proteins that usually accumulate in these conditions were not detectable soon after, and spores lacking different heat-shock proteins exhibited identical wet heat resistance to wild-type spores, suggesting that they have no effect per se on spore resistance (Movahedi and Waites 2000; Melly and Setlow 2001; Movahedi and Waites 2002). In contrast to heat resistance parameters, the zT-value, i.e. the temperature elevation causing a 10-fold reduction in D-values, remains relatively constant (from 8°C to 12°C), as shown for instance for B. cereus and for B. licheniformis spores (Raso et al.1995; Sala et al.1995; González et al.1999). Both the chemist energy of activation Ea and (food) microbiologist zT are expressing the effects of changes in temperatures on the inactivation reaction; zT may even be derived from Ea by a simple calculation (Hoxey, Thomas and Davies 2007). This conserved zT value suggests that the basic mechanism of spore inactivation by wet heat remains constant whatever the sporulation temperature is, despite structural changes in spores and variable efficiency of spore heat killing. Figure 3. View largeDownload slide Variability in the resistance to heat of spores of B. subtilis (), B. cereus (), and of other Bacillus sp. () as a function of temperature of sporulation, composition of the sporulation medium (carbohydrates and mineral compounds, pH and aw), method of spore preparation (agar, broth, biofilm) and of other factors (ethanol, heat and pH shock, oxygen availability). In each considered paper, the resistance to heat is expressed by decimal reduction time D, time to 2 or 3 decimal reductions, or number of decimal reduction after a given time of heat-treatment. A value of 1 () has been arbitrarily attributed to the parameter expressing the lowest resistance to heat reported. Data are available in Table S1 (Supporting Information). Data have been extracted from Elbisi and Ordal (1956), El-Bisi and Ordal (1956), Amaha and Ordal (1957), Lechowich and Ordal (1962), Fleming and Ordal (1964), Levinson and Hyatt (1964), Lundgren (1967), Khoury, Lombardi and Slepecky (1987), Lindsay et al. (1990), Condon, Bayarte and Sala (1992), De Pieri and Ludlow (1992), Raso et al. (1995), Sala et al. (1995), Mazas et al. (1997), González et al. (1999), Movahedi and Waites (2000), Atrih and Foster (2001), Cazemier, Wagenaars and ter Steeg (2001), Melly et al. (2002), Movahedi and Waites (2002), Lee et al. (2003), de Vries et al. (2005), Rose et al. (2007), Baweja et al. (2008), Nguyen Thi Minh, Perrier-Cornet and Gervais (2008), Mazas et al. (2009), Stecchini et al. (2009), Garcia, van der Voort and Abee (2010), Baril et al. (2011), Nguyen Thi Minh et al. (2011), Planchon et al. (2011), Baril et al. (2012), Olivier, Bull and Chapman (2012), van der Voort and Abee (2013), Abbas et al. (2014), Bressuire-Isoard et al. (2016) and Hayrapetyan, Abee and Nierop Groot (2016). Figure 3. View largeDownload slide Variability in the resistance to heat of spores of B. subtilis (), B. cereus (), and of other Bacillus sp. () as a function of temperature of sporulation, composition of the sporulation medium (carbohydrates and mineral compounds, pH and aw), method of spore preparation (agar, broth, biofilm) and of other factors (ethanol, heat and pH shock, oxygen availability). In each considered paper, the resistance to heat is expressed by decimal reduction time D, time to 2 or 3 decimal reductions, or number of decimal reduction after a given time of heat-treatment. A value of 1 () has been arbitrarily attributed to the parameter expressing the lowest resistance to heat reported. Data are available in Table S1 (Supporting Information). Data have been extracted from Elbisi and Ordal (1956), El-Bisi and Ordal (1956), Amaha and Ordal (1957), Lechowich and Ordal (1962), Fleming and Ordal (1964), Levinson and Hyatt (1964), Lundgren (1967), Khoury, Lombardi and Slepecky (1987), Lindsay et al. (1990), Condon, Bayarte and Sala (1992), De Pieri and Ludlow (1992), Raso et al. (1995), Sala et al. (1995), Mazas et al. (1997), González et al. (1999), Movahedi and Waites (2000), Atrih and Foster (2001), Cazemier, Wagenaars and ter Steeg (2001), Melly et al. (2002), Movahedi and Waites (2002), Lee et al. (2003), de Vries et al. (2005), Rose et al. (2007), Baweja et al. (2008), Nguyen Thi Minh, Perrier-Cornet and Gervais (2008), Mazas et al. (2009), Stecchini et al. (2009), Garcia, van der Voort and Abee (2010), Baril et al. (2011), Nguyen Thi Minh et al. (2011), Planchon et al. (2011), Baril et al. (2012), Olivier, Bull and Chapman (2012), van der Voort and Abee (2013), Abbas et al. (2014), Bressuire-Isoard et al. (2016) and Hayrapetyan, Abee and Nierop Groot (2016). Other growth conditions also have a significant impact on spore resistance to wet heat (Fig. 3; Supporting Information Table S1), but with no clear explanation and link with the spore structure so far. Bacillussubtilis spores produced at low aw were less resistant to wet heat than those formed at high aw (Nguyen Thi Minh, Perrier-Cornet and Gervais 2008). Bacillusanthracis spores formed at acid pH were found to be more resistant to wet heat than spores formed at alkaline and neutral pH (Baweja et al.2008). The alkaline pH may increase mineralization of spores during sporulation further contributing to heat resistance. Spores produced under low aeration or anaerobic conditions have a higher resistance to heat than spores produced under standard oxygen condition (Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Abbas et al.2014). Mineralization of the sporulation media has a marked influence on the wet heat resistance of spores, with a correlative effect on the water content of the spore core. Indeed the supplementation of sporulation media or the remineralization with Ca2+of spores demineralized by acid treatments generally tend to increase spore resistance to wet heat of species such as B. subtilis, B. megaterium, B. anthracis or B. licheniformis (Amaha and Ordal 1957; Cazemier, Wagenaars and ter Steeg 2001; Igura et al.2003; Baweja et al.2008; Nguyen Thi Minh et al.2011). However how mineralization affects the water content of the spore core is still poorly understood. Ca2+ enrichment within the CaDPA complex may limit the mobility of water molecules in the spore core (Setlow 2006). Bacilluscereus spores formed in a medium containing a high glutamate concentration had a higher heat resistance than spores formed in presence of low glutamate concentration (de Vries et al.2005). While the chemical composition was not affected, resistance to wet heat of B. cereus spores increased as sporulation medium viscosity increased (Stecchini et al.2009), and was higher for B. subtilis spores formed on agar than in broth (Rose et al.2007). The latter spores formed in broth or on agar exhibited no differences in DPA, core water content and α/β-type SASPs, despite being significantly different in their wet heat resistance (Rose et al.2007). Sporulation conditions and resistance to physical and chemical agents other than heat In addition to wet heat resistance, the effect of sporulation conditions on resistance to a range of chemical and physical agents has also been investigated. The properties of spores affected by the sporulation conditions are very diverse, as the sporulation conditions influencing spore lead to modifications of properties (Table 1). However, for a given sporulation condition, the different resistance properties are independently modified. For instance, increasing B. subtilis sporulation temperature from 22°C to 48°C had a tremendous effect on wet heat resistance (D values increased more than 10-fold) but no detectable effect on dry heat resistance and only a marginal effect on resistance to formaldehyde and some other DNA-damaging chemicals (Melly et al.2002; Cortezzo and Setlow 2005). This could be explained by similar concentrations of α/β-type SASPs, which are known to be major determinants of resistance to dry heat of spores formed at diverse temperatures (Melly et al.2002). Table 1. Effects of sporulation conditions on the resistance of Bacillus sp. spores to physical and chemical agents other than wet heat. Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) a logR: number of log-reduction after a given treatment. b F10: Fluence for a 10-fold reduction of the spore population. c LD90: or D, time to kill 90% of the spore population. View Large Table 1. Effects of sporulation conditions on the resistance of Bacillus sp. spores to physical and chemical agents other than wet heat. Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) a logR: number of log-reduction after a given treatment. b F10: Fluence for a 10-fold reduction of the spore population. c LD90: or D, time to kill 90% of the spore population. View Large The mechanisms underlying the observed effects of sporulation conditions on spore resistance to various chemical and physical agents are generally not well understood, largely due to the diversity of the tested sporulation conditions and inactivation treatments and often to the absence of structural, biochemical or molecular characterizations of spores. Supplementing the sporulation media with thioproline, cysteine or cystine resulted in significant increases in B. subtilis spore resistance to H2O2 and solar UV radiation (290–400 nm) but not to 254-nm UV radiation (Moeller et al.2011). This could be related to the potentially radioprotective effects of these amino acids against reactive oxygen species generated by H2O2 and solar UV radiation. Moreover, de-coated spores lost this enhanced resistance, suggesting that amino acid uptake enhances the protective role of the spore coat (Moeller et al.2011). Penetration of chemicals into the spore core, and therefore the permeability of its surrounding layers, may be critical for sensitivity to DNA-damaging agents. Spores of B. subtilis formed in liquid media were more sensitive to nitrous acid and super-oxidized water than spores formed on solid media (Rose et al.2007). Similarly, B. subtilis spores formed at lower temperatures were more sensitive to nitrous acid (Cortezzo and Setlow 2005) and they showed a higher rate of methylamine uptake, suggesting a more permeable spore inner membrane. Higher reactivity to deleterious chemicals in the spore core could also be due to higher water content, as observed especially in spores formed at low sporulation temperatures. However, the authors noted that even the significant differences in fatty acid profiles and core water contents were rather small and seem unlikely to be the only cause of the observed effects (Cortezzo and Setlow 2005). Other structures may be implicated, for instance the spore coat, as spore coat elimination somehow increased the sensitivity of spores to nitrous acid (Cortezzo and Setlow 2005). Impact of sporulation environment on spore germination The sporulation environment also influences spore germination and outgrowth after exposure to favorable conditions. Spore germination is usually triggered by (i) nutrients, including sugars, purine nucleosides and amino acids, (ii) non-nutrient agents such as CaDPA, surfactants or dodecylamine or (iii) physical treatments such as hyperbaric treatment (Paidhungat et al.2002; Setlow 2014a). Bacillussubtilis spores formed in a nutrient-poor sporulation medium germinated more slowly in response to L-valine and to a mixture of L-asparagine, D-glucose, D-fructose and K+ (AGFK) than spores formed in a nutrient-rich medium (Ramirez-Peralta et al.2012). This effect was likely due to a lower level of the germinant receptor proteins and the lipoprotein GerD required for efficient B. subtilis germination with L-alanine observed in the spores formed in the nutrient-poor medium (Mongkolthanaruk, Robinson and Moir 2009; Ramirez-Peralta et al.2012). Bacilluscereus spores produced in a medium enriched in amino acids and glucose showed enhanced germination, together with increased levels of expression of the seven ger operons (Hornstra et al.2006). Changes in expression of ger operons caused variations in the number of germinant receptor proteins in the spore, and overexpression of gerA operons in B. subtilis also led to faster germination (Cabrera-Martinez et al.2003). Similarly, B. subtilis spores germinated more efficiently in response to moderate high pressure (150 MPa) when formed in a rich rather than in a poor nutrient medium (Doona et al.2014). Germination at this pressure is known to be germinant receptor-dependent (Setlow 2003), and a higher level of Ger proteins was observed in the spores formed in a nutrient-rich medium than in a poor one. In contrast, the germination of both B. subtilis and B. weihenstephanensis spores at very high pressure (i.e. >500 MPa), which is not germinant receptor-dependent, was not affected by sporulation temperature (Garcia, van der Voort and Abee 2010; Doona et al.2014). Modulation of the quantity and/or activity of two exosporium enzymes, i.e. alanine racemase and the nucleoside hydrolase, may also affect nutrient-driven germination (Todd et al.2003; de Vries et al.2005). Alanine racemase converts L-alanine into D-alanine, which inhibits spore germination, while nucleotide hydrolase degrades inosine, a major germinant for spores of the B. cereus group. Among other factors, sporulation temperature certainly has a major effect on spore germination (Table 2). However, high germination rate or high germination efficiency has been associated to either high or low sporulation temperatures depending on strain, germinant, and conditions tested. In independent experiments for instance, sporulation temperature had opposite effects on the sensitivity of B. weihenstephanensis KBAB4 spores to L-alanine alone or in combination with inosine (Table 2; Garcia, van der Voort and Abee 2010; Planchon et al.2011). Table 2. Variations in germination of Bacillus spores according to sporulation conditions. Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) View Large Table 2. Variations in germination of Bacillus spores according to sporulation conditions. Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) View Large Germination, mainly characterized by spore rehydration (revealed by the spore transformation from phase-bright to phase-dark) and loss of resistance, is only the very first stage in the process leading to the initiation of the first cell division and settlement of a daughter population. The question of whether later post-germination stages of spore evolution (duration of outgrowth, or time to first cell division for instance) are affected by sporulation conditions has not been thoroughly studied. Modification of the sporulation environment triggers deep metabolic and physiological adaptations that cause structural modifications in sporulating cells and alter spore properties. When related to vegetative cells, including from Bacillus sp., these adaptations create measurable effects on lag times during growth, and these effects have been extensively documented (Swinnen et al.2004). However, this question has received less attention in relation to spores. Sporulation at low aw, low temperature or alkaline pH favored colony formation of B. subtilis spores on a nutrient agar at aw close to the limit of growth (Nguyen Thi Minh et al.2011). However, absence of colony formation can signify impaired germination or may equally signify impaired adaptation of germinated and/or outgrowing cells. With specific regard to outgrowth capacity, B. weihenstephanensis spores formed at different temperatures showed no significant change at a range of incubation temperatures, despite sharp modifications of spore germination and resistance to several stresses (Garcia, van der Voort and Abee 2010). Post-formation exposure of spores to non-lethal temperatures for several days resulted in different patterns of germination and outgrowth, associated to different levels of ribosomal RNA or proteins (Segev, Smith and Ben-Yehuda 2012; Segev et al.2013). There is no evidence that such differences can be obtained by modifying sporulation conditions, but differences in the ‘molecular cargo’ gathered within spores can significantly affect revival. Is there really a spore ‘memory’, i.e. molecular or physical traces created during sporulation that will influence outgrowth and further growth events following germination? This question receives an increasing attention. For instance, alanine-induced outgrowth of B. subtilis spores is dependent on the production during sporulation and on accumulation within spores of alanine dehydrogenase, a metabolic enzyme that converts alanine to pyruvate. More generally, this memory may vary within the progeny of a sporulating cell population, and therefore could be beneficial to survive diverse selection pressures in fluctuating environments (Mutlu et al.2018). CONCLUSIONS Sporulation in the environment occurs in very diverse ecological niches (Carlin 2011; Gauvry et al.2017). Bacterial spores will inexorably contaminate food-industry environments, healthcare facilities and other microbiologically-sensitive sites, where their resistance and recovery capacities will be largely unknown and practically unpredictable. Meanwhile, applications involving spore-forming bacteria are increasing and require spores with reliable properties. Consequently, understanding how sporulation in natural or industrial environments shapes spore properties is vital in order to design efficient microbial elimination or growth control strategies or, in contrast, to optimize the requisite properties for applications in human and animal health, crop protection, and other industrial domains. In addition, individual spores in populations show heterogeneous behaviors in response to heat treatment or to suboptimal recovery environments (Eijlander, Abee and Kuipers 2011; Pandey et al.2013; van Melis et al.2014; Warda et al.2015). Whether and how spore properties can affect this variable pattern of behavior warrants investigation in order to help better predict spores' fate, and thus potentially improve important processes such as ensuring food safety and quality. Laboratory studies show that sporulation media have a strong impact on spore properties, and that sporulation temperature is definitely one of the most important environmental factors influencing bacterial spore behavior. However, we cannot claim that lab studies provide a suitable description of the conditions occurring in the environment. Surveys on the ecology of spore-forming bacteria may contribute to help anticipate and identify spatial and temporal distributions in terms of places and times when sporulation really occurs. In other words, does spore formation occur at low level in very different environments or as bursts at highly specific locations and/or during or after specific and possibly rare meteorological events? This question was addressed in a review on spore-forming and thermophilic Geobacillus sp., but despite multiple hypotheses, no fully satisfactory answer has emerged (Zeigler 2014). Can we also infer the primary cause of differences in Bacillus spore resistance or germination from modifications in spore structure and composition? Spore properties are usually clearly modified by environmental conditions, but the link with structural modifications or changes in spore composition is less than clear and likely complex. Some processes are deeply multifactorial, such as germination which is a cascade of molecular and biochemical events. We cannot expect to identify sensitive spore biomarkers to reliably predict spore properties and avoid costly and time-consuming phenotypic characterization (of resistance, of germination and recovery, and so on) anytime soon. However, we can anticipate greater support for the identification of biomarkers through the development of analytical tools. Raman spectroscopy or fatty acid profile analysis, for instance, enabled a fine discrimination of laboratory conditions (media) in which B. cereus spores were formed (Ehrhardt et al.2010; Dettman et al.2015). The omics techniques will also help decipher the complex network of regulations (Bate, Bonneau and Eichenberger 2014; Abhyankar et al.2017) that ultimately determines the desirable or undesirable properties of bacterial spores entering the world of human activities. FUNDING The PhD contract of CBI was supported by Montpellier University (France) and Doctoral School GAIA. Conflicts of interest. None declared. REFERENCES Abbas AA , Planchon S , Jobin M et al. Absence of oxygen affects the capacity to sporulate and the spore properties of Bacillus cereus . Food Microbiol 2014 ; 42 : 122 – 31 . Google Scholar CrossRef Search ADS PubMed Abhyankar W , Pandey R , Ter Beek A et al. Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation . 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Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms

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Oxford University Press
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© FEMS 2018.
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0168-6445
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1574-6976
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10.1093/femsre/fuy021
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Abstract

Abstract Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health and industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties. resistance, germination, sporulation environment, structure, exosporium, coat INTRODUCTION Spores are forms of resistance of Bacillus sp. and other Firmicutes. The elimination of bacterial spores of pathogenic species in healthcare facilities remains a problematic issue that hampers efforts to prevent nosocomial infections (Bottone 2010; Maillard 2011; Barra-Carrasco and Paredes-Sabja 2014). The thermal intensity of food-processing operations (in the canning industry or in ultra-high-temperature processing) is also designed to efficiently inactivate bacterial spores. Even so, spores may still survive, which means many unprocessed or processed foods depend on in-storage refrigeration for bacterial safety or prevention of spoilage (Logan 2012; Wells-Bennik et al.2016). That said, spore-forming bacteria also have many desirable properties (Wolken, Tramper and van der Werf 2003). Their vegetative and/or sporulating cells produce enzymes and metabolites that are exploited for applications in agronomics, veterinary medicine, healthcare and biotech. For example Bacillusthuringiensis is in widespread use for the bioinsecticidal properties of toxins it forms during sporulation and Bacillus sp. are also exploited as plant-growth promoters or bio-fertilizers (Perez-Garcia, Romero and de Vicente 2011). At industrial scale, spore resistance favors the formulation and storage of commercial products at ambient temperature for extended periods with only limited effects on spore viability. Regarding animal health, Bacillus probiotics are used in humans as dietary supplements and in livestock, poultry or aquaculture as growth or disease resistance promoters and competitive exclusion agents (Cutting 2011). Probiotic spores are also attractive delivery vehicle for oral vaccination thanks to resistance to gastric acidity (Rosales-Mendoza, Angulo and Meza 2016). Bacterial spores that borrow genomic roots from the Firmicutes phylum are the most resistant form of life on Earth. Spores can remain dormant and metabolically inert for very long periods and longevity and lifetimes of hundreds to thousands year have been reported in several instances (Nicholson 2003; Gould 2006). Spores can be dispersed by wind, water, or living hosts, and by transportation of natural or industrial material to locations far away from the sporulation site, and ultimately in an environment not immediately suitable for growth. For example, the presence of thermophilic spores in cold environments can be attributed to dispersion from geothermal aquifers and hydrothermal vents (de Rezende et al.2013). When the environment becomes favorable, spores can break dormancy and re-initiate a lifecycle through germination and outgrowth processes. This lifecycle represents a successful mechanism for widespread dispersal of spore-forming bacteria on Earth. Thus spores are found in highly diverse environmental niches, from abiotic and biotic fractions of soil including the rhizosphere to the gut of terrestrial and aquatic animals including mammals and on to industrial installations and healthcare facilities. They thus are exposed to a huge diversity of adverse conditions, from extreme temperature swings, eventually freezing and thawing, to physical abrasion, desiccation or exposure to corrosive chemicals, solar or industrial radiation, or even predation (Nicholson et al.2000). The sporulation process generates a spore that has a radically different structure to the vegetative cell (Fig. 1). The outermost ‘balloon-like’ layer, called exosporium, is found in some Bacillus species, such as Bacillus cereus or Bacillus anthracis. This first point of spore contact with the environment is highly hydrophobic and allows the spores to adhere to cells and abiotic surfaces (Oliva, Turnbough and Kearney 2009; Lequette et al.2011; Xue et al.2011; Stewart 2015). Bacillus subtilis has no exosporium—its outermost structure is a proteic ‘crust’ (Henriques and Moran 2007; Imamura et al.2010; McKenney et al.2010; Leggett et al.2012; McKenney, Driks and Eichenberger 2013). The B. subtilis spore coat is a protein-rich structure composed of an inner and outer layer separated in species possessing an exosporium by a large ‘interspace’ (Giorno et al.2007; Leggett et al.2012; Setlow 2014a,b). The relative permeability, to lysozyme for instance, of the outer membrane beneath the spore coat does not likely confer resistance to spores (Setlow 2006). The peptidoglycan of the spore cortex is required for the maintenance of spore core dehydration and also plays a role in dormancy (Foster and Popham 2002). The inner membrane that protects the spore core is characterized by low permeability to small molecules and water (Paredes-Sabja and Sarker 2011; Bassi, Cappa and Cocconcelli 2012). The spore core contains DNA encased in small acid-soluble spore proteins (SASP), high levels of dipicolinic acid (DPA) chelated to divalent Ca2+ cations (CaDPA), and minerals, and is characterized by a low water content (Setlow 2014a,b). The assembly and final arrangement of these different spore structures confers survival and persistence through resistance and dormancy, pending suitable conditions for growth. Figure 1. View largeDownload slide Structure of a Bacillus cereus spore and roles of the spore components in resistance. (A) Negative staining image, (B) transmission electron microscopy image of thin section and (C) schematic view of a B. cereus spore structure. The exosporium (Ex) is a balloon-like structure loosely anchored to the coat through protein-protein interactions. The exosporium contributes to spore attachment to abiotic and biotic surfaces. Spore appendages (Sa) are clearly visible on panel (A). The coat (Ct) is separated from the exosporium by an interspace (Is). The coat represents a large part of total spore proteins, organized as a permeability barrier to degradative enzymes, and detoxifies deleterious chemicals. The coat protects the innermost spore components and maintains low water permeability. The outer membrane (Om) may accumulate carotenoids, pigments protecting the spore against UV radiations. The cortex (Cx) is made of peptidoglycan. Its role in resistance is unknown, but it may be implicated in the low water content of the spore core. The low permability of the inner membrane (Im) contributes to the protection against disinfectants and some DNA damaging chemicals. The core (Co) contains the DNA saturated with α/β type SASP protecting against UV- and γ-radiation, dry heat and wet heat, genotoxic chemicals and some oxidizing agents. Spore core has a low water content, a high level of DPA and divalent metals protecting against desiccation, dry and moist heat. Photographic images are from INRA Avignon, France. Adapted from Driks (1999), Nicholson et al. (2000), Melly et al. (2002), Setlow et al. (2006), Setlow (2014b), Stewart (2015) and Knudsen et al. (2016). Figure 1. View largeDownload slide Structure of a Bacillus cereus spore and roles of the spore components in resistance. (A) Negative staining image, (B) transmission electron microscopy image of thin section and (C) schematic view of a B. cereus spore structure. The exosporium (Ex) is a balloon-like structure loosely anchored to the coat through protein-protein interactions. The exosporium contributes to spore attachment to abiotic and biotic surfaces. Spore appendages (Sa) are clearly visible on panel (A). The coat (Ct) is separated from the exosporium by an interspace (Is). The coat represents a large part of total spore proteins, organized as a permeability barrier to degradative enzymes, and detoxifies deleterious chemicals. The coat protects the innermost spore components and maintains low water permeability. The outer membrane (Om) may accumulate carotenoids, pigments protecting the spore against UV radiations. The cortex (Cx) is made of peptidoglycan. Its role in resistance is unknown, but it may be implicated in the low water content of the spore core. The low permability of the inner membrane (Im) contributes to the protection against disinfectants and some DNA damaging chemicals. The core (Co) contains the DNA saturated with α/β type SASP protecting against UV- and γ-radiation, dry heat and wet heat, genotoxic chemicals and some oxidizing agents. Spore core has a low water content, a high level of DPA and divalent metals protecting against desiccation, dry and moist heat. Photographic images are from INRA Avignon, France. Adapted from Driks (1999), Nicholson et al. (2000), Melly et al. (2002), Setlow et al. (2006), Setlow (2014b), Stewart (2015) and Knudsen et al. (2016). Many spore-forming species sporulate in laboratory conditions. The range of media and incubation environments that support sporulation is huge, but how far does it influence spore properties like resistance? This question addresses fundamental issues of how intensively metabolic, physiological or molecular adaptations during growth will interfere with sporulation and final spore properties. But it also has practical implications: many applications, such as risk assessment in foods, analysis of disinfectant efficiency, probiotics for health, crop pest control, or design of bioindicators, use spores that are usually formed in artificial environments. How reliable are inactivation, survival, germination and growth predictions for food safety or hygiene of healthcare facilities based on cultivated spores compared to spores formed in natural environments? How do sporulation conditions impact the gastric survival of spores administered as probiotics and their interactions with the gut microbiota and mucosa? The aim of this review is to illustrate how sporulation conditions affect the spore properties of Bacillus sp., and to identify, when possible, the most plausible molecular and physiological mechanisms behind the observed effects. Environmental conditions influence sporulation efficiency, spore structure and composition Factors modulating spore formation In response to nutrient limitation and quorum sensing signals, vegetative cells of Bacillus species transform into sporulating cells after an asymmetric division through a complex developmental process. A phosphorelay regulatory system activates the sporulation pathway through transcription factors that are phosphorylated by sensor kinases (Sonenshein 2000; Hilbert and Piggot 2004; Higgins and Dworkin 2012; Tan and Ramamurthi 2014; Decker and Ramamurthi 2017). Sporulation yield can be estimated as the amount of vegetative cells that undergo a complete sporulation process. Spores are enumerated by plate-counting heat-resistant cells in a suspension, or by counting refractile cells with a phase-contrast microscope or a flow cytometer. Unsurprisingly, the environmental factors that influence growth of Bacillus cells also deeply affect sporulation. To the best of our knowledge, there is still no evidence that Bacillus spores can be formed outside the range of temperature, pH and water activity (aw) allowing growth. Sporulation yield is usually maximal at optimal growth temperature, pH or aw, and decreases as temperature, pH or aw stray from optimum, which tends to lengthen the sporulation process (Mazas et al.1997; Baweja et al.2008; Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Garcia, van der Voort and Abee 2010; Nguyen Thi Minh et al.2011; Planchon et al.2011; Baril et al.2012). For instance, the time to complete growth and sporulation of B. subtilis ATCC31324 is 3 days at 37°C, pH 8.0 and high aw in a standard nutrient broth (optimal conditions). It increases to 10 days at 45°C and 14 days at 19°C, to 20 days at pH 6.0 or pH 10.0, and to 17 days at aw = 0.950 (Nguyen Thi Minh et al.2011). Observations on Bacillus weihenstephanensis and Bacillus licheniformis suggested that temperature and pH could have the same quantitative influence on both maximum specific growth rate and sporulation rate (Baril et al.2012). Counts of Bacillus sp. spores were generally maximal at optimal temperature, pH and aw and remained high in a wide range of culture conditions (Mazas et al.1997; Baweja et al.2008; Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Garcia, van der Voort and Abee 2010; Nguyen Thi Minh et al.2011). Sporulation yield of the psychrotrophic B. weihenstephanensis KBAB4, which has a minimal temperature for growth of approximately 6°C, was >99% at 12°C and at 30°C (Garcia, van der Voort and Abee 2010), whereas spore formation was also observed at 10°C and 7°C but with much lower efficiency. Inhibition of B. subtilis sporulation by high salinity (about 7% NaCl) occurs at an early stage due to impaired activity of the response regulator Spo0A governing entry into sporulation and of the alternate sigma factor σH (Widderich et al.2016). In many instances, total spore count and sporulation yield remain relatively unchanged as conditions approach the limits of sporulation and growth. However, sporulation, which can be high within a large range of conditions allowing growth, varies strongly with strain, sporulation media and incubation conditions. Sporulation yields for B. thuringiensis and B. cereus, for example, are affected by oxygen concentration: the quantity of spores formed (and cell growth) were lower under oxygen limitation than under aerobiosis (Avignone-Rossa, Arcas and Mignone 1992; Boniolo et al.2012; Abbas et al.2014). The sporulation medium's level of nutrients, in particularly mono/di-valent cations, is also a major factor. Supplementation with Mn2+, Mg2+, Zn2+, Ca2+ increased the sporulation yield of Bacillus species and improved the stability of spores which no longer underwent spontaneous germination (Atrih and Foster 2001). Time to complete full sporulation of B. subtilis was almost five times longer in absence than in presence of Ca2+ (Nguyen Thi Minh et al.2011). It has long been known that the sporulation process and final spore yield are amino acid- and carbohydrate-dependent (Schaeffer et al.1965). The combined effects of yeast extract, peptone and glucose enhanced the spore yield of B. megaterium (Verma et al.2013). Likewise, the addition of glucose and ribose in the sporulation medium increased the spore yield of B. subtilis and B. cereus (Warriner and Waites 1999; de Vries et al.2005; Monteiro et al.2005). Optimizing glucose and Mg2+concentration in a sporulation medium led to a 17-fold increase in spore yield of a B. subtilis strain promoting plant growth (Posada-Uribe, Romero-Tabarez and Villegas-Escobar 2015). The mechanism by which cell metabolism affects sporulation is complex. For instance, the sporulation process of B. cereus ATCC14579 was substantially longer in presence of high glutamate concentration (de Vries et al.2005). However, different glutamate concentrations had no effect on the temporal expression of sigF and sigG genes encoding the key transcriptional sporulation factors σF and σG. Glutamate concentration may therefore affect the programming of sporulation events that occur after those directly controlled by σG, which include mother cell lysis and spore maturation. Factors influencing spore structure and composition Spore structure and composition are also affected by changes in the sporulation environment (Fig. 2). Spores accumulate minerals in the spore core (mainly Ca2+, Mg2+ and Mn2+) during the sporulation process. Spore-core mineral concentrations vary widely and are highly dependent on the composition of the sporulation medium (Bassi, Cappa and Cocconcelli 2012). The spore core is also characterized by a high content of CaDPA that may form, according to a recently proposed model, an inorganic polymer bridged by water molecules that maintains the spore core in an as-yet undetermined state described as glassy or gel-like (Setlow and Li 2015). This water-CaDPA polymer favors spore resistance by immobilizing proteins or molecules such as membrane lipids (Cowan et al.2003; Cowan et al.2004). Bacillussubtilis spores lacking the ability to synthesize DPA and that are formed in a growth medium without added DPA have a much higher core water content than spores formed in a DPA-supplemented medium (Paidhungat et al.2000). Spores of several Bacillus have a lower water content when formed on nutrient agar with a mix of metal cations (Ca2+, Mg2+, Fe2+, K+ and Mn2+) than with Mn2+ only or without cations (Cazemier, Wagenaars and ter Steeg 2001; Nguyen Thi Minh et al.2011). Sporulation temperature is also among the factors that have the strongest effect on DPA and core water content, although this effect may not be systematically reported (Melly et al.2002; Kaieda et al.2013). DPA concentrations were higher in B. cereus and B. anthracis spores formed at high temperatures (30°C and 45°C, respectively) rather than at low temperatures (10°C and 25°C, respectively) (Baweja et al.2008; Planchon et al.2011). Water content was lower in B. subtilis spores formed at high temperature than at low temperature (Beaman and Gerhardt 1986; Melly et al.2002; Nguyen Thi Minh et al.2011). Moreover higher sporulation temperature has also been correlated with higher levels of core spore mineralization (Palop, Sala and Condon 1999; Igura et al.2003). The cortex peptidoglycan of spores prepared at different temperatures showed subtle changes in structure, with slightly increased percentages of cross-linked muramic acid in spores prepared at high temperatures (Melly et al.2002). Cortex peptidoglycan structure was also substantially modified in spores of various Bacillus species produced in a nutrient-poor medium without any carbon source compared to a rich medium (Atrih and Foster 2001). The authors hypothesized that a change in muropeptide ratio, as well as a probable decrease in number of muropeptides containing δ-lactam, reveals a functional defect of the cortex biosynthesis pathway and/or maturation machinery. Likewise, addition of MnCl2 to sporulation media resulted in an altered peptidoglycan composition and peptidoglycan chain cross-linking. Mn2+ may therefore affect the expression of genes and/or the activity of enzymes involved in cortex biosynthesis (Atrih and Foster 2001). Figure 2. View largeDownload slide A synthetic view on the major factors known to influence structure and composition of Bacillus spores. indicates an effect on a spore component or structure. Effect on a spore component or structure with consequences on resistance to heat is indicated by , with consequences on resistance to chemical biocides by , and with consequences on spore germination by . Coat P, coat proteins. Ger, germinant receptors. DPA, dipicolinic acid. M+, metal ions. H2O, spore core water content. Ex, exosporium. Is, interspace. Ct, coat. Cx, cortex. Im, Om, inner and outer membrane. Co, core. Figure 2. View largeDownload slide A synthetic view on the major factors known to influence structure and composition of Bacillus spores. indicates an effect on a spore component or structure. Effect on a spore component or structure with consequences on resistance to heat is indicated by , with consequences on resistance to chemical biocides by , and with consequences on spore germination by . Coat P, coat proteins. Ger, germinant receptors. DPA, dipicolinic acid. M+, metal ions. H2O, spore core water content. Ex, exosporium. Is, interspace. Ct, coat. Cx, cortex. Im, Om, inner and outer membrane. Co, core. Variations in sporulation temperature, pH or concentration of inorganic salts resulted in substantial variations in spore volumes, which ranged from 0.38 μm3; to 0.79 μm3; for B. cereus and 0.53 μm3; to 0.71 μm3; for B. megaterium (Zhou et al.2017). Bacillussubtilis spores were nearly twice as small when formed in a Ca2+-deprived sporulation medium (Nguyen Thi Minh et al.2011). Spores of several B. cereus strains produced in liquid media were significantly smaller than the ones formed on agar or in biofilms (van der Voort and Abee 2013). The roughness of the spore surface can be affected by incubation temperature or by aw of the sporulation medium (Nguyen Thi Minh et al.2011). Spore swelling (shrinking) in response to high (low) relative humidity and core (de)hydration of B. thuringiensis and B. subtilis spores could suggest a link between spore (de)hydration and spore size (Westphal et al.2003; Sunde et al.2009). However, spore hydration is not the only cause: varying sporulation conditions led to marked differences in spore volumes but without any difference in spore wet density (Zhou et al.2017). Other structural modifications are directly observable with electron microscopy. Examples are that B. cereus exosporium is damaged and detached when spores are formed at high temperature (Faille et al.2007), or that the outer coat of B. subtilis spores is thicker in spores formed in a chemically-defined broth than in a rich agar medium (Abhyankar et al.2016). Differences in coat protein profiles were observed for B. subtilis spores prepared at various temperatures or in broth vs. agar plates (Melly et al.2002; Rose et al.2007; Abhyankar et al.2016). In contrast α/β-type SASP remained unaffected in the same sporulation conditions (Melly et al.2002; Rose et al.2007). The intensity of the electrophoretic bands of the coat proteins CotA, CotG, CotB and CotS was lower in extracts from spores prepared at 22°C and 30°C than at 48°C. Several proteins of coat and exosporium extracts from B. cereus differed in spores formed at 20°C or 37°C (Bressuire-Isoard et al.2016). Among these, the CotE protein was proportionally greater in extracts from spores produced at 20°C than at 37°C. These differences may result from differences in the amount and/or extractability of the proteins. In B. subtilis for instance, protein extraction yielded a higher amount of total proteins from spores prepared on agar than from spores prepared in broth (Rose et al.2007). Nevertheless, relative coat protein contents did not show any major difference between spores formed in agar vs. in broth (Driks 1999; Rose et al.2007). Bacilluscereus and B. subtilis spores produced at different sporulation temperatures showed significant differences in fatty acid (FA) composition. Total amount of anteiso FA increased in B. cereus spores produced at low temperature (Planchon et al.2011). The anteiso-to-iso ratio and the amount of unsaturated FA in B. subtilis spore inner membrane increased as temperature decreased (Cortezzo and Setlow 2005). These changes correspond to those generally observed in Bacillus spp. cells during low-temperature adaptation (Diomande et al.2015) and likely reflect a need to maintain membrane fluidity. A similar higher anteiso-to-iso FA ratio in the inner membrane was observed for spores formed on plates vs. broth (Rose et al.2007). Spore structure, organization and composition are clearly very sensitive to changes in the physical and chemical sporulation environment. Sporulation conditions affect spore resistance Many spore structures are involved in spore resistance Resistance to extreme temperatures is a distinguishing property between bacterial spores and vegetative cells, as wet-heat inactivation of spores requires a roughly 45°C higher temperature than wet-heat inactivation of vegetative cells (Setlow 2014a,b; Checinska, Paszczynski and Burbank 2015). Spore killing by wet heat is mainly due to damages to core proteins and denaturation of enzymes involved in metabolism (Coleman et al.2007; Setlow 2014a,b; Wells-Bennik et al.2016). Resistance to wet heat involves DNA saturation with α/β-type SASP proteins, low core water, high DPA and mineral content, likely reducing molecular mobility in the core and protecting proteins against thermal denaturation and irreversible aggregation (Sunde et al.2009; Setlow 2014a,b). The spore cortex, through the level of peptidoglycan cross-linking, could also be involved in maintaining the dehydrated state of B. subtilis spore cores (Atrih and Foster 1999; Driks 1999). However, how such modifications in cortex composition actually maintain spore core dehydration remains unknown. Increase in resistance during spore maturation concomitantly with cross-linking of the outer coat proteins has recently suggested a possible role of coat in resistance to wet heat (Sanchez-Salas et al.2011; Abhyankar et al.2015). Spores of at least several Bacillus sp. share different mechanisms for resistance to dry heat and wet heat. Resistance to dry heat is related to DNA saturation by α/β-type SASP in the core and activation of systems to repair dry heat-induced DNA damage during spore outgrowth like the RecA protein (Setlow and Setlow 1996; Nicholson et al.2000; Setlow 2014a,b; Setlow et al.2014). Besides resistance to heat, spores are also resistant to an array of physical insults including desiccation, freeze–thaw cycles, UV and γ-radiation, high hydrostatic pressure and chemical insults involving a variety of toxic effects (Nicholson et al.2000; Setlow 2014a,b; Checinska, Paszczynski and Burbank 2015). Resistance to UV involves two major factors—α/β-type SASP binding to DNA, and DNA repair during spore outgrowth—plus minor factors such as carotenoids in spores outer layers, and low water and high DPA content in spore core (Setlow 2014a,b). Spores are 10–50 times more resistant to UV radiation than vegetative cells, depending on the species studied (Nicholson et al.2000). Strong acid treatments, organic solvents at high temperatures, and oxidizing agents such as hydrogen peroxide all cause major damages in the inner spore membrane, where oxidation of membrane proteins may result in rupture and cell death (Cortezzo et al.2004; Cortezzo and Setlow 2005). Alkali treatments mainly inactivate the lytic enzymes of the coat that hydrolyze the cortex during germination (Setlow et al.2002). The spore coat is the first line of defense against large molecules targeting the spore cortex, and it plays a major role in shielding the spore against oxidizing agents such as chlorine dioxide, hypochlorite or peroxynitrite (Setlow 2000; Genest et al.2002; Melly et al.2002; Young and Setlow 2003). Resistance to chemicals involves a large number of factors, such as detoxifying enzymes of spore coat and/or exosporium including catalase or superoxide dismutase, low permeability of the spore inner membrane, DNA protection by α/β-type SASPs and delete DNA repair systems (Setlow 2006; Setlow 2014a,b). Factors influencing resistance to heat The impact of environmental conditions on spore heat resistance was already observed back in the late 1920s on B. anthracis spores, which were more heat-resistant when formed at 37°C than at 18°C (Williams 1929). Spores of Bacillus sp. prepared at suboptimal temperatures are consistently less resistant to wet heat than spores prepared at near-optimal temperatures (Beaman and Gerhardt 1986; Condon, Bayarte and Sala 1992; Raso et al.1995; González et al.1999; Baweja et al.2008; Garcia, van der Voort and Abee 2010; Baril et al.2011; Nguyen Thi Minh et al.2011; Planchon et al.2011; Bressuire-Isoard et al.2016). The difference can be quite significant. Heat-resistance parameters such as decimal reduction times, or D values, can vary by a factor greater than 10 depending on sporulation temperature (Fig. 3; Table S1, Supporting Information), and some authors have even proposed the concept of an optimal sporulation temperature and pH at which spores get their maximal wet-heat resistance (Baril et al.2012; Trunet et al.2015). Spore core water content decreases as sporulation temperature increases and, as stated earlier under ‘Factors influencing spore structure and composition’, there is a reciprocal positive correlation between core dehydration and spore wet heat resistance. DPA contributes to spore wet heat resistance by replacing water molecules and therefore maintaining low spore core water content. Similarly, a higher sporulation temperature correlated with higher levels of spore mineralization which results in higher heat resistance (Palop, Sala and Condon 1999). Sporulation temperature has subtle effects on the cortex structure of B. subtilis spores, with potential changes in core hydration (Melly et al.2002). Even a few minutes heat-shock or cold-shock at an appropriate time during sporulation can affect wet heat resistance. However, heat-shock and cold-shock proteins that usually accumulate in these conditions were not detectable soon after, and spores lacking different heat-shock proteins exhibited identical wet heat resistance to wild-type spores, suggesting that they have no effect per se on spore resistance (Movahedi and Waites 2000; Melly and Setlow 2001; Movahedi and Waites 2002). In contrast to heat resistance parameters, the zT-value, i.e. the temperature elevation causing a 10-fold reduction in D-values, remains relatively constant (from 8°C to 12°C), as shown for instance for B. cereus and for B. licheniformis spores (Raso et al.1995; Sala et al.1995; González et al.1999). Both the chemist energy of activation Ea and (food) microbiologist zT are expressing the effects of changes in temperatures on the inactivation reaction; zT may even be derived from Ea by a simple calculation (Hoxey, Thomas and Davies 2007). This conserved zT value suggests that the basic mechanism of spore inactivation by wet heat remains constant whatever the sporulation temperature is, despite structural changes in spores and variable efficiency of spore heat killing. Figure 3. View largeDownload slide Variability in the resistance to heat of spores of B. subtilis (), B. cereus (), and of other Bacillus sp. () as a function of temperature of sporulation, composition of the sporulation medium (carbohydrates and mineral compounds, pH and aw), method of spore preparation (agar, broth, biofilm) and of other factors (ethanol, heat and pH shock, oxygen availability). In each considered paper, the resistance to heat is expressed by decimal reduction time D, time to 2 or 3 decimal reductions, or number of decimal reduction after a given time of heat-treatment. A value of 1 () has been arbitrarily attributed to the parameter expressing the lowest resistance to heat reported. Data are available in Table S1 (Supporting Information). Data have been extracted from Elbisi and Ordal (1956), El-Bisi and Ordal (1956), Amaha and Ordal (1957), Lechowich and Ordal (1962), Fleming and Ordal (1964), Levinson and Hyatt (1964), Lundgren (1967), Khoury, Lombardi and Slepecky (1987), Lindsay et al. (1990), Condon, Bayarte and Sala (1992), De Pieri and Ludlow (1992), Raso et al. (1995), Sala et al. (1995), Mazas et al. (1997), González et al. (1999), Movahedi and Waites (2000), Atrih and Foster (2001), Cazemier, Wagenaars and ter Steeg (2001), Melly et al. (2002), Movahedi and Waites (2002), Lee et al. (2003), de Vries et al. (2005), Rose et al. (2007), Baweja et al. (2008), Nguyen Thi Minh, Perrier-Cornet and Gervais (2008), Mazas et al. (2009), Stecchini et al. (2009), Garcia, van der Voort and Abee (2010), Baril et al. (2011), Nguyen Thi Minh et al. (2011), Planchon et al. (2011), Baril et al. (2012), Olivier, Bull and Chapman (2012), van der Voort and Abee (2013), Abbas et al. (2014), Bressuire-Isoard et al. (2016) and Hayrapetyan, Abee and Nierop Groot (2016). Figure 3. View largeDownload slide Variability in the resistance to heat of spores of B. subtilis (), B. cereus (), and of other Bacillus sp. () as a function of temperature of sporulation, composition of the sporulation medium (carbohydrates and mineral compounds, pH and aw), method of spore preparation (agar, broth, biofilm) and of other factors (ethanol, heat and pH shock, oxygen availability). In each considered paper, the resistance to heat is expressed by decimal reduction time D, time to 2 or 3 decimal reductions, or number of decimal reduction after a given time of heat-treatment. A value of 1 () has been arbitrarily attributed to the parameter expressing the lowest resistance to heat reported. Data are available in Table S1 (Supporting Information). Data have been extracted from Elbisi and Ordal (1956), El-Bisi and Ordal (1956), Amaha and Ordal (1957), Lechowich and Ordal (1962), Fleming and Ordal (1964), Levinson and Hyatt (1964), Lundgren (1967), Khoury, Lombardi and Slepecky (1987), Lindsay et al. (1990), Condon, Bayarte and Sala (1992), De Pieri and Ludlow (1992), Raso et al. (1995), Sala et al. (1995), Mazas et al. (1997), González et al. (1999), Movahedi and Waites (2000), Atrih and Foster (2001), Cazemier, Wagenaars and ter Steeg (2001), Melly et al. (2002), Movahedi and Waites (2002), Lee et al. (2003), de Vries et al. (2005), Rose et al. (2007), Baweja et al. (2008), Nguyen Thi Minh, Perrier-Cornet and Gervais (2008), Mazas et al. (2009), Stecchini et al. (2009), Garcia, van der Voort and Abee (2010), Baril et al. (2011), Nguyen Thi Minh et al. (2011), Planchon et al. (2011), Baril et al. (2012), Olivier, Bull and Chapman (2012), van der Voort and Abee (2013), Abbas et al. (2014), Bressuire-Isoard et al. (2016) and Hayrapetyan, Abee and Nierop Groot (2016). Other growth conditions also have a significant impact on spore resistance to wet heat (Fig. 3; Supporting Information Table S1), but with no clear explanation and link with the spore structure so far. Bacillussubtilis spores produced at low aw were less resistant to wet heat than those formed at high aw (Nguyen Thi Minh, Perrier-Cornet and Gervais 2008). Bacillusanthracis spores formed at acid pH were found to be more resistant to wet heat than spores formed at alkaline and neutral pH (Baweja et al.2008). The alkaline pH may increase mineralization of spores during sporulation further contributing to heat resistance. Spores produced under low aeration or anaerobic conditions have a higher resistance to heat than spores produced under standard oxygen condition (Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Abbas et al.2014). Mineralization of the sporulation media has a marked influence on the wet heat resistance of spores, with a correlative effect on the water content of the spore core. Indeed the supplementation of sporulation media or the remineralization with Ca2+of spores demineralized by acid treatments generally tend to increase spore resistance to wet heat of species such as B. subtilis, B. megaterium, B. anthracis or B. licheniformis (Amaha and Ordal 1957; Cazemier, Wagenaars and ter Steeg 2001; Igura et al.2003; Baweja et al.2008; Nguyen Thi Minh et al.2011). However how mineralization affects the water content of the spore core is still poorly understood. Ca2+ enrichment within the CaDPA complex may limit the mobility of water molecules in the spore core (Setlow 2006). Bacilluscereus spores formed in a medium containing a high glutamate concentration had a higher heat resistance than spores formed in presence of low glutamate concentration (de Vries et al.2005). While the chemical composition was not affected, resistance to wet heat of B. cereus spores increased as sporulation medium viscosity increased (Stecchini et al.2009), and was higher for B. subtilis spores formed on agar than in broth (Rose et al.2007). The latter spores formed in broth or on agar exhibited no differences in DPA, core water content and α/β-type SASPs, despite being significantly different in their wet heat resistance (Rose et al.2007). Sporulation conditions and resistance to physical and chemical agents other than heat In addition to wet heat resistance, the effect of sporulation conditions on resistance to a range of chemical and physical agents has also been investigated. The properties of spores affected by the sporulation conditions are very diverse, as the sporulation conditions influencing spore lead to modifications of properties (Table 1). However, for a given sporulation condition, the different resistance properties are independently modified. For instance, increasing B. subtilis sporulation temperature from 22°C to 48°C had a tremendous effect on wet heat resistance (D values increased more than 10-fold) but no detectable effect on dry heat resistance and only a marginal effect on resistance to formaldehyde and some other DNA-damaging chemicals (Melly et al.2002; Cortezzo and Setlow 2005). This could be explained by similar concentrations of α/β-type SASPs, which are known to be major determinants of resistance to dry heat of spores formed at diverse temperatures (Melly et al.2002). Table 1. Effects of sporulation conditions on the resistance of Bacillus sp. spores to physical and chemical agents other than wet heat. Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) a logR: number of log-reduction after a given treatment. b F10: Fluence for a 10-fold reduction of the spore population. c LD90: or D, time to kill 90% of the spore population. View Large Table 1. Effects of sporulation conditions on the resistance of Bacillus sp. spores to physical and chemical agents other than wet heat. Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) Variable conditions during sporulation Species Physical or chemical agent Consequences on resistance Reference Temperature B. subtilis Glutaraldehyde at 9 g L−1 logRa = 3 after < 15 min for spores produced at 22°C. After 45 min for spores produced at 48°C Melly et al. (2002) B. subtilis Sterilox at 240 ml L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. logR = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis H2O2 at 5% (v/v) After 30 min logR> 3 for spores produced at 22°C. After 45 min logR ± 2 for spores produced at 48°C. Melly et al. (2002) B. subtilis Sterilox at 240 mL L−1 free chlorine After 15 min logR ± 2 for spores produced at 22°C. log R = 0 for spores produced at 49°C Melly et al. (2002) B. subtilis Betadine at 85% After 1 min logR> 3 for spores produced at 22°C. After 4 min logR ± 1 for spores produced at 48°C Melly et al. (2002) B. subtilis High pressure (300 Mpa for 60 min and at 55°C) logR = 2 for spores prepared at 30°C; logR = 4 for spores prepared at 37°C or 44°C Igura et al. (2003) B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higher for spores formed at 25°C than for spores formed at 46°C Tovar-Rojo et al. (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. logR<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 gL−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Hypochlorite at 2.5 g L−1 After 15 min logR> 3 for spores produced at 25°C. log R<1 for spores produced at 46°C. Young and Setlow (2003) B. subtilis Chloride dioxide at 2 g L−1 After 10 min logR = 4 for spores produced at 25°C and logR±0 for spores produced at 46°C. Young and Setlow (2003) B. subtilis High pressure (800 MPa and 70°C) logR = 2.5 for spores prepared at 30°C logR = 4 for spores prepared at 44°C; logR = 4 for spores prepared at 48°C Margosch et al. (2004) B. subtilis Atmospheric plasma D = 27 s for spores formed at 22°C D; = 65 s for spores formed at 47°C Deng et al. (2005) B. weihenstephanenis NaOH 1M After 90 min logR = 0.5 for spores produced at 30°C and logR = 2.5 for for spores produced at 10°C Planchon et al. (2011) B. weihenstephanenis Pulsed-UV light. Fluence = 0.7 J cm−2 logR = 2 for spores produced at 30°C and logR = 4.5 for spores produced at 10°C Planchon et al. (2011) Composition of the sporulation medium B. subtilis UV in the range 280–400 nm or 320–400 nm Increasing F10b for spores prepared in media supplemented with cysteine, cysteine or thioproline Moeller et al. (2011) B. subtilis H2O2 5% LD90c> 28 min for spores prepared in media supplemented with cysteine, cysteine or thioproline. LD90 = 13 for control. Moeller et al. (2011) Temperature and composition of the sporulation agar or broth B. sporothermodurans and B. amyloliquefaciens (B. coagulans) High pressure (500 MPa) and temperature (110°C) 1.5–6 fold decrease in Dc-values when sporulation temperature increased from 30°C–37°C (37°C–50°C). 10-fold decrease to 2.5 increase in D with mineralization of the sporulation medium and sporulation temperature increase Olivier, Bull and Chapman (2012) Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced in anaerobiosis and logR = 2.5 in aerobiosis Abbas et al. (2014) pH B. subtilis 35% H2O2 D = 140 s for spores prepared at ph = 7.0; D = 75 at pH = 8.5 Eschlbeck, Bauer and Kulozik (2017) pH, temperature B. subtilis High pressure at 350 MPa for 60 min and 40°C logR = 1.8 for spores formed in standard conditions. logR = 3.4 (0.7) for spores at pH 6.0 (10); logR = 3.4 for spores formed at 19°C Nguyen Thi Minh et al. (2011) Spore preparation method B. subtilis Nitrous acid at 400 mmol L−1 or super-oxidized water Lag time in inactivation curves longer for spores formed on agar plates longer than in broth Rose et al. (2007) a logR: number of log-reduction after a given treatment. b F10: Fluence for a 10-fold reduction of the spore population. c LD90: or D, time to kill 90% of the spore population. View Large The mechanisms underlying the observed effects of sporulation conditions on spore resistance to various chemical and physical agents are generally not well understood, largely due to the diversity of the tested sporulation conditions and inactivation treatments and often to the absence of structural, biochemical or molecular characterizations of spores. Supplementing the sporulation media with thioproline, cysteine or cystine resulted in significant increases in B. subtilis spore resistance to H2O2 and solar UV radiation (290–400 nm) but not to 254-nm UV radiation (Moeller et al.2011). This could be related to the potentially radioprotective effects of these amino acids against reactive oxygen species generated by H2O2 and solar UV radiation. Moreover, de-coated spores lost this enhanced resistance, suggesting that amino acid uptake enhances the protective role of the spore coat (Moeller et al.2011). Penetration of chemicals into the spore core, and therefore the permeability of its surrounding layers, may be critical for sensitivity to DNA-damaging agents. Spores of B. subtilis formed in liquid media were more sensitive to nitrous acid and super-oxidized water than spores formed on solid media (Rose et al.2007). Similarly, B. subtilis spores formed at lower temperatures were more sensitive to nitrous acid (Cortezzo and Setlow 2005) and they showed a higher rate of methylamine uptake, suggesting a more permeable spore inner membrane. Higher reactivity to deleterious chemicals in the spore core could also be due to higher water content, as observed especially in spores formed at low sporulation temperatures. However, the authors noted that even the significant differences in fatty acid profiles and core water contents were rather small and seem unlikely to be the only cause of the observed effects (Cortezzo and Setlow 2005). Other structures may be implicated, for instance the spore coat, as spore coat elimination somehow increased the sensitivity of spores to nitrous acid (Cortezzo and Setlow 2005). Impact of sporulation environment on spore germination The sporulation environment also influences spore germination and outgrowth after exposure to favorable conditions. Spore germination is usually triggered by (i) nutrients, including sugars, purine nucleosides and amino acids, (ii) non-nutrient agents such as CaDPA, surfactants or dodecylamine or (iii) physical treatments such as hyperbaric treatment (Paidhungat et al.2002; Setlow 2014a). Bacillussubtilis spores formed in a nutrient-poor sporulation medium germinated more slowly in response to L-valine and to a mixture of L-asparagine, D-glucose, D-fructose and K+ (AGFK) than spores formed in a nutrient-rich medium (Ramirez-Peralta et al.2012). This effect was likely due to a lower level of the germinant receptor proteins and the lipoprotein GerD required for efficient B. subtilis germination with L-alanine observed in the spores formed in the nutrient-poor medium (Mongkolthanaruk, Robinson and Moir 2009; Ramirez-Peralta et al.2012). Bacilluscereus spores produced in a medium enriched in amino acids and glucose showed enhanced germination, together with increased levels of expression of the seven ger operons (Hornstra et al.2006). Changes in expression of ger operons caused variations in the number of germinant receptor proteins in the spore, and overexpression of gerA operons in B. subtilis also led to faster germination (Cabrera-Martinez et al.2003). Similarly, B. subtilis spores germinated more efficiently in response to moderate high pressure (150 MPa) when formed in a rich rather than in a poor nutrient medium (Doona et al.2014). Germination at this pressure is known to be germinant receptor-dependent (Setlow 2003), and a higher level of Ger proteins was observed in the spores formed in a nutrient-rich medium than in a poor one. In contrast, the germination of both B. subtilis and B. weihenstephanensis spores at very high pressure (i.e. >500 MPa), which is not germinant receptor-dependent, was not affected by sporulation temperature (Garcia, van der Voort and Abee 2010; Doona et al.2014). Modulation of the quantity and/or activity of two exosporium enzymes, i.e. alanine racemase and the nucleoside hydrolase, may also affect nutrient-driven germination (Todd et al.2003; de Vries et al.2005). Alanine racemase converts L-alanine into D-alanine, which inhibits spore germination, while nucleotide hydrolase degrades inosine, a major germinant for spores of the B. cereus group. Among other factors, sporulation temperature certainly has a major effect on spore germination (Table 2). However, high germination rate or high germination efficiency has been associated to either high or low sporulation temperatures depending on strain, germinant, and conditions tested. In independent experiments for instance, sporulation temperature had opposite effects on the sensitivity of B. weihenstephanensis KBAB4 spores to L-alanine alone or in combination with inosine (Table 2; Garcia, van der Voort and Abee 2010; Planchon et al.2011). Table 2. Variations in germination of Bacillus spores according to sporulation conditions. Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) View Large Table 2. Variations in germination of Bacillus spores according to sporulation conditions. Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) Variable conditions during sporulation Species Germination inducer Effects on germination Reference Temperature B. anthracis L-alanine at 4 mmol L−1 Faster germination of spores formed at 45°C than at 20°C Baweja et al. (2008) B. cereus High hydrostatic pressure The ungerminated fraction of spores prepared at 20°C was higher after HHP treatment in the range 250–700 MPa than for spores prepared at 37°C Raso, Barbosa-Canovas and Swanson (1998a,b) B. cereus Inosine and L-alanine Spores formed at 15°C–20°C are more sensitive and show a higher germination rate than at 37°C Gounina-Allouane, Broussolle and Carlin (2008) B. subtilis Dodecylamine > 90% germination within 50 min for spores prepared at 23°C and < 25% for spores prepared at 44°C Cortezzo and Setlow (2005) B. subtilis High hydrostatic pressure at 500 MPa > 90% germination in 2.5 min for spores prepared at 23°C and 30°C; <50% for spores prepared at 44°C Black et al. (2007) B. subtilis L-valine 10 mM or 10 mmol l−1 (each) AGFK Rate of germination of spores prepared at 37°C or 43°C lower than of spores prepared at 23°C or 30°C Luu et al. (2015) B. weihenstephanensis 12.5 mmol L−1 inosine and 25 mmol L−1 L-alanine Faster germination with spores formed at 37°C than at 12°C or 20°C Garcia, van der Voort and Abee (2010) B. weihenstephanenis High hydrostatic pressure Higher germination of spores formed at 20°C and 37°C under 150 MPa; no difference under 500 Mpa Garcia, van der Voort and Abee (2010) B. weihenstephanenis L-alanine at different concentrations Spores formed at 10°C are more sensitive to L-alanine and show a higher germination rate than at 30°C Planchon et al. (2011) Composition of the sporulation agar or broth B. megaterium Diverse nutrient germinants Germination rate and spore activation requirement depending on sporulation medium. Higher germination rates for spores prepared with minerals and CaCl2 supplementation Levinson and Hyatt (1964) B. megaterium L-alanine or inosine No effect of heat-shock on germination of spores formed on citrate-based medium. With acetate, heat-shock improves germination Hitchins, Slepecky and Greene (1972) Composition of the sporulation agar or broth B. cereus L-alanine 10 mmol L−1 or inosine 5 mmol L−1 High glutamate concentration favors germination de Vries et al. (2005) Spore preparation method B. subtilis Dodecylamine Slower germination of spores prepared on agar than on liquid Setlow, Cowan and Setlow 2003; Rose et al. (2007) Spore preparation method B. cereus L-alanine 5 mmol L−1 and inosine 2.5 mmol L−1 Spores formed in biofilms showed a germination capacity lower than spores formed in broth van der Voort and Abee (2013) Spore preparation method B. subtilis Moderate hydrostatic pressure at 150 MPa Lower germination rate of spores formed in a poor medium Doona et al. (2014) NaCl concentration B. subtilis High hydrostatic pressure at 500 MPa 75% germination in 3 min for spores prepared without NaCl addition; <25% for spores prepared with 1 mol l−1 NaCl Black et al. (2007) NaCl, CaCl2 B. cereus 1 mmol L−1 L-alanine Inhibition of germination in spores prepared with 1 M NaCl; Germination > control (No NaCl and CaCl2) for spores prepared with 1 M NaCl and 0.05 M CaCl2 Fleming and Ordal (1964) Oxygen availability B. cereus L-alanine at different concentrations > 90% (< 50%) germination for spores prepared in anaerobiosis (aerobiosis) Abbas et al. (2014) View Large Germination, mainly characterized by spore rehydration (revealed by the spore transformation from phase-bright to phase-dark) and loss of resistance, is only the very first stage in the process leading to the initiation of the first cell division and settlement of a daughter population. The question of whether later post-germination stages of spore evolution (duration of outgrowth, or time to first cell division for instance) are affected by sporulation conditions has not been thoroughly studied. Modification of the sporulation environment triggers deep metabolic and physiological adaptations that cause structural modifications in sporulating cells and alter spore properties. When related to vegetative cells, including from Bacillus sp., these adaptations create measurable effects on lag times during growth, and these effects have been extensively documented (Swinnen et al.2004). However, this question has received less attention in relation to spores. Sporulation at low aw, low temperature or alkaline pH favored colony formation of B. subtilis spores on a nutrient agar at aw close to the limit of growth (Nguyen Thi Minh et al.2011). However, absence of colony formation can signify impaired germination or may equally signify impaired adaptation of germinated and/or outgrowing cells. With specific regard to outgrowth capacity, B. weihenstephanensis spores formed at different temperatures showed no significant change at a range of incubation temperatures, despite sharp modifications of spore germination and resistance to several stresses (Garcia, van der Voort and Abee 2010). Post-formation exposure of spores to non-lethal temperatures for several days resulted in different patterns of germination and outgrowth, associated to different levels of ribosomal RNA or proteins (Segev, Smith and Ben-Yehuda 2012; Segev et al.2013). There is no evidence that such differences can be obtained by modifying sporulation conditions, but differences in the ‘molecular cargo’ gathered within spores can significantly affect revival. Is there really a spore ‘memory’, i.e. molecular or physical traces created during sporulation that will influence outgrowth and further growth events following germination? This question receives an increasing attention. For instance, alanine-induced outgrowth of B. subtilis spores is dependent on the production during sporulation and on accumulation within spores of alanine dehydrogenase, a metabolic enzyme that converts alanine to pyruvate. More generally, this memory may vary within the progeny of a sporulating cell population, and therefore could be beneficial to survive diverse selection pressures in fluctuating environments (Mutlu et al.2018). CONCLUSIONS Sporulation in the environment occurs in very diverse ecological niches (Carlin 2011; Gauvry et al.2017). Bacterial spores will inexorably contaminate food-industry environments, healthcare facilities and other microbiologically-sensitive sites, where their resistance and recovery capacities will be largely unknown and practically unpredictable. Meanwhile, applications involving spore-forming bacteria are increasing and require spores with reliable properties. Consequently, understanding how sporulation in natural or industrial environments shapes spore properties is vital in order to design efficient microbial elimination or growth control strategies or, in contrast, to optimize the requisite properties for applications in human and animal health, crop protection, and other industrial domains. In addition, individual spores in populations show heterogeneous behaviors in response to heat treatment or to suboptimal recovery environments (Eijlander, Abee and Kuipers 2011; Pandey et al.2013; van Melis et al.2014; Warda et al.2015). Whether and how spore properties can affect this variable pattern of behavior warrants investigation in order to help better predict spores' fate, and thus potentially improve important processes such as ensuring food safety and quality. Laboratory studies show that sporulation media have a strong impact on spore properties, and that sporulation temperature is definitely one of the most important environmental factors influencing bacterial spore behavior. However, we cannot claim that lab studies provide a suitable description of the conditions occurring in the environment. Surveys on the ecology of spore-forming bacteria may contribute to help anticipate and identify spatial and temporal distributions in terms of places and times when sporulation really occurs. In other words, does spore formation occur at low level in very different environments or as bursts at highly specific locations and/or during or after specific and possibly rare meteorological events? This question was addressed in a review on spore-forming and thermophilic Geobacillus sp., but despite multiple hypotheses, no fully satisfactory answer has emerged (Zeigler 2014). Can we also infer the primary cause of differences in Bacillus spore resistance or germination from modifications in spore structure and composition? Spore properties are usually clearly modified by environmental conditions, but the link with structural modifications or changes in spore composition is less than clear and likely complex. Some processes are deeply multifactorial, such as germination which is a cascade of molecular and biochemical events. We cannot expect to identify sensitive spore biomarkers to reliably predict spore properties and avoid costly and time-consuming phenotypic characterization (of resistance, of germination and recovery, and so on) anytime soon. However, we can anticipate greater support for the identification of biomarkers through the development of analytical tools. 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Journal

FEMS Microbiology ReviewsOxford University Press

Published: Sep 1, 2018

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