sporulation environment influences spore properties in bacillus

13
FEMS Microbiology Reviews, fuy021, 42, 2018, 614–626 doi: 10.1093/femsre/fuy021 Advance Access Publication Date: 17 May 2018 Review Article REVIEW ARTICLE Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms Christelle Bressuire-Isoard, V ´ eronique Broussolle and Fr ´ ed´ eric Carlin UMR408 SQPOV “S´ ecurit ´ e et Qualit ´ e des Produits d’Origine V ´ eg´ etale”, INRA–Avignon Universit´ e, Centre de Recherche PACA, CS40509, Site Agroparc, 84914 Avignon Cedex 9, France Corresponding author: INRA, Centre de Recherche PACA, 228 Route de l’A´ erodrome, CS40509, Domaine Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9, France. Tel: +33(0)4 32 72 25 19; E-mail: [email protected] One sentence summary: The environment in which spores of Bacillus sp form shapes spore properties and capacities of resistance, germination and further growth. Editor: Oscar Kuipers 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. Keywords: resistance; germination; sporulation environment; structure; exosporium; coat INTRODUCTION Spores are forms of resistance of Bacillus sp. and other Fir- micutes. 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, Received: 28 September 2017; Accepted: 16 May 2018 C FEMS 2018. All rights reserved. For permissions, please e-mail: [email protected] 614 Downloaded from https://academic.oup.com/femsre/article/42/5/614/4998855 by guest on 24 January 2022

Upload: khangminh22

Post on 20-Jan-2023

1 views

Category:

Documents


0 download

TRANSCRIPT

FEMS Microbiology Reviews, fuy021, 42, 2018, 614–626

doi: 10.1093/femsre/fuy021Advance Access Publication Date: 17 May 2018Review Article

REVIEW ARTICLE

Sporulation environment influences spore propertiesin Bacillus: evidence and insights on underlyingmolecular and physiological mechanismsChristelle Bressuire-Isoard, Veronique Broussolle and Frederic Carlin∗

UMR408 SQPOV “Securite et Qualite des Produits d’Origine Vegetale”, INRA–Avignon Universite, Centre deRecherche PACA, CS40509, Site Agroparc, 84914 Avignon Cedex 9, France∗Corresponding author: INRA, Centre de Recherche PACA, 228 Route de l’Aerodrome, CS40509, Domaine Saint-Paul, Site Agroparc, 84914 AvignonCedex 9, France. Tel: +33(0)4 32 72 25 19; E-mail: [email protected] sentence summary: The environment in which spores of Bacillus sp form shapes spore properties and capacities of resistance, germination andfurther growth.Editor: Oscar Kuipers

ABSTRACT

Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health andindustry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Sporeresistance and dormancy are important properties for applications in medicine, veterinary health, food safety, cropprotection and other domains. The resistance of bacterial spores results from a protective multilayered structure and fromthe unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking ofdormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims toillustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in whichspores form. We look at the physiological and molecular mechanisms underpinning how sporulation media andenvironment 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 andprocess efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulationconditions influence spore properties.

Keywords: resistance; germination; sporulation environment; structure; exosporium; coat

INTRODUCTION

Spores are forms of resistance of Bacillus sp. and other Fir-micutes. The elimination of bacterial spores of pathogenicspecies in healthcare facilities remains a problematic issuethat hampers efforts to prevent nosocomial infections (Bottone2010; Maillard 2011; Barra-Carrasco and Paredes-Sabja 2014).The thermal intensity of food-processing operations (in thecanning 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 orprocessed foods depend on in-storage refrigeration for bacterialsafety or prevention of spoilage (Logan 2012; Wells-Benniket al. 2016). That said, spore-forming bacteria also have manydesirable properties (Wolken, Tramper and van der Werf 2003).Their vegetative and/or sporulating cells produce enzymes andmetabolites that are exploited for applications in agronomics,

Received: 28 September 2017; Accepted: 16 May 2018C© FEMS 2018. All rights reserved. For permissions, please e-mail: [email protected]

614

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 615

veterinary medicine, healthcare and biotech. For exampleBacillus thuringiensis is in widespread use for the bioinsecticidalproperties 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 industrialscale, spore resistance favors the formulation and storage ofcommercial products at ambient temperature for extendedperiods with only limited effects on spore viability. Regardinganimal health, Bacillus probiotics are used in humans as dietarysupplements and in livestock, poultry or aquaculture as growthor disease resistance promoters and competitive exclusionagents (Cutting 2011). Probiotic spores are also attractive deliv-ery vehicle for oral vaccination thanks to resistance to gastricacidity (Rosales-Mendoza, Angulo and Meza 2016).

Bacterial spores that borrow genomic roots from the Firmi-cutes phylum are themost resistant form of life on Earth. Sporescan remain dormant and metabolically inert for very long peri-ods and longevity and lifetimes of hundreds to thousands yearhave been reported in several instances (Nicholson 2003; Gould2006). Spores can be dispersed by wind, water, or living hosts,and by transportation of natural or industrial material to loca-tions far away from the sporulation site, and ultimately in anenvironment not immediately suitable for growth. For exam-ple, the presence of thermophilic spores in cold environmentscan be attributed to dispersion from geothermal aquifers andhydrothermal vents (de Rezende et al. 2013). When the envi-ronment becomes favorable, spores can break dormancy andre-initiate a lifecycle through germination and outgrowth pro-cesses. This lifecycle represents a successful mechanism forwidespread dispersal of spore-forming bacteria on Earth. Thusspores are found in highly diverse environmental niches, fromabiotic and biotic fractions of soil including the rhizosphere tothe gut of terrestrial and aquatic animals including mammalsand on to industrial installations and healthcare facilities. Theythus are exposed to a huge diversity of adverse conditions, fromextreme temperature swings, eventually freezing and thawing,to physical abrasion, desiccation or exposure to corrosive chem-icals, solar or industrial radiation, or even predation (Nicholsonet al. 2000).

The sporulation process generates a spore that has a radi-cally different structure to the vegetative cell (Fig. 1). The out-ermost ‘balloon-like’ layer, called exosporium, is found in someBacillus species, such as Bacillus cereus or Bacillus anthracis. Thisfirst point of spore contact with the environment is highly hy-drophobic and allows the spores to adhere to cells and abi-otic surfaces (Oliva, Turnbough and Kearney 2009; Lequetteet al. 2011; Xue et al. 2011; Stewart 2015). Bacillus subtilis has noexosporium—its outermost structure is a proteic ‘crust’ (Hen-riques and Moran 2007; Imamura et al. 2010; McKenney et al.2010; Leggett et al. 2012; McKenney, Driks and Eichenberger2013). The B. subtilis spore coat is a protein-rich structure com-posed of an inner and outer layer separated in species possess-ing 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 thespore coat does not likely confer resistance to spores (Setlow2006). The peptidoglycan of the spore cortex is required for themaintenance of spore core dehydration and also plays a rolein dormancy (Foster and Popham 2002). The inner membranethat protects the spore core is characterized by low permeabil-ity to smallmolecules andwater (Paredes-Sabja and Sarker 2011;Bassi, Cappa andCocconcelli 2012). The spore core contains DNAencased in small acid-soluble spore proteins (SASP), high lev-els of dipicolinic acid (DPA) chelated to divalent Ca2+ cations(CaDPA), and minerals, and is characterized by a low water con-

Co

Ex

Ex

Cx

Ct

(A) (B)

(C)

Is

Sa

0.5 µm

0.2 µm

Figure 1. Structure of a Bacillus cereus spore and roles of the spore componentsin resistance. (A) Negative staining image, (B) transmission electron microscopyimage 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 throughprotein-protein interactions. The exosporium contributes to spore attachmentto 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). Thecoat represents a large part of total spore proteins, organized as a permeabilitybarrier to degradative enzymes, and detoxifies deleterious chemicals. The coatprotects the innermost spore components and maintains low water permeabil-

ity. The outer membrane (Om) may accumulate carotenoids, pigments protect-ing the spore against UV radiations. The cortex (Cx) is made of peptidoglycan. Itsrole in resistance is unknown, but it may be implicated in the low water contentof the spore core. The low permability of the inner membrane (Im) contributes

to the protection against disinfectants and some DNA damaging chemicals. Thecore (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 oxidiz-ing agents. Spore core has a low water content, a high level of DPA and divalent

metals protecting against desiccation, dry and moist heat. Photographic imagesare from INRAAvignon, France. Adapted fromDriks (1999), Nicholson et al. (2000),Melly et al. (2002), Setlow et al. (2006), Setlow (2014b), Stewart (2015) and Knudsen

et al. (2016).

tent (Setlow 2014a,b). The assembly and final arrangement ofthese different spore structures confers survival and persistencethrough resistance and dormancy, pending suitable conditionsfor growth.

Many spore-forming species sporulate in laboratory condi-tions. The range of media and incubation environments thatsupport sporulation is huge, but how far does it influence sporeproperties like resistance? This question addresses fundamen-tal issues of how intensively metabolic, physiological or molec-ular adaptations during growth will interfere with sporulationand final spore properties. But it also has practical implications:many applications, such as risk assessment in foods, analysis ofdisinfectant efficiency, probiotics for health, crop pest control,or design of bioindicators, use spores that are usually formed inartificial environments. How reliable are inactivation, survival,germination and growth predictions for food safety or hygieneof healthcare facilities based on cultivated spores compared tospores formed in natural environments? How do sporulationconditions impact the gastric survival of spores administeredas probiotics and their interactions with the gut microbiota andmucosa? The aim of this review is to illustrate how sporulationconditions affect the spore properties of Bacillus sp., and to iden-tify, when possible, themost plausiblemolecular and physiolog-ical mechanisms behind the observed effects.

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

616 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

Environmental conditions influence sporulationefficiency, spore structure and composition

Factors modulating spore formationIn response to nutrient limitation and quorum sensing signals,vegetative cells of Bacillus species transform into sporulatingcells after an asymmetric division through a complex devel-opmental process. A phosphorelay regulatory system activatesthe sporulation pathway through transcription factors that arephosphorylated by sensor kinases (Sonenshein 2000; Hilbert andPiggot 2004; Higgins and Dworkin 2012; Tan and Ramamurthi2014; Decker and Ramamurthi 2017). Sporulation yield can beestimated as the amount of vegetative cells that undergo acomplete sporulation process. Spores are enumerated by plate-counting heat-resistant cells in a suspension, or by counting re-fractile cells with a phase-contrastmicroscope or a flow cytome-ter.

Unsurprisingly, the environmental factors that influencegrowth of Bacillus cells also deeply affect sporulation. To the bestof our knowledge, there is still no evidence that Bacillus sporescan be formed outside the range of temperature, pH and wateractivity (aw) allowing growth. Sporulation yield is usually max-imal at optimal growth temperature, pH or aw, and decreasesas temperature, pH or aw stray from optimum, which tendsto lengthen the sporulation process (Mazas et al. 1997; Bawejaet 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 timeto complete growth and sporulation of B. subtilis ATCC31324 is3 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 daysat 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 Bacillusweihenstephanensis and Bacillus licheniformis suggested that tem-perature and pH could have the same quantitative influence onboth maximum specific growth rate and sporulation rate (Barilet al. 2012). Counts of Bacillus sp. spores were generally maximalat optimal temperature, pH and aw and remained high in a widerange of culture conditions (Mazas et al. 1997; Baweja et al. 2008;Nguyen Thi Minh, Perrier-Cornet and Gervais 2008; Garcia, vander Voort and Abee 2010; Nguyen Thi Minh et al. 2011). Sporu-lation yield of the psychrotrophic B. weihenstephanensis KBAB4,which has a minimal temperature for growth of approximately6◦C, was >99% at 12◦C and at 30◦C (Garcia, van der Voort andAbee 2010), whereas spore formation was also observed at10◦C and 7◦C but with much lower efficiency. Inhibition of B.subtilis sporulation by high salinity (about 7% NaCl) occurs at anearly stage due to impaired activity of the response regulatorSpo0A governing entry into sporulation and of the alternatesigma factor σH (Widderich et al. 2016). In many instances, totalspore count and sporulation yield remain relatively unchangedas conditions approach the limits of sporulation and growth.However, sporulation, which can be high within a large rangeof conditions allowing growth, varies strongly with strain,sporulation media and incubation conditions. Sporulationyields for B. thuringiensis and B. cereus, for example, are affectedby oxygen concentration: the quantity of spores formed (andcell growth) were lower under oxygen limitation than underaerobiosis (Avignone-Rossa, Arcas and Mignone 1992; Bonioloet al. 2012; Abbas et al. 2014). The sporulation medium’s levelof nutrients, in particularly mono/di-valent cations, is also amajor factor. Supplementation with Mn2+, Mg2+, Zn2+, Ca2+

increased the sporulation yield of Bacillus species and improvedthe stability of spores which no longer underwent spontaneous

germination (Atrih and Foster 2001). Time to complete fullsporulation of B. subtiliswas almost five times longer in absencethan in presence of Ca2+ (Nguyen Thi Minh et al. 2011). It haslong been known that the sporulation process and final sporeyield are amino acid- and carbohydrate-dependent (Schaefferet al. 1965). The combined effects of yeast extract, peptoneand glucose enhanced the spore yield of B. megaterium (Vermaet al. 2013). Likewise, the addition of glucose and ribose in thesporulation medium increased the spore yield of B. subtilis andB. cereus (Warriner andWaites 1999; de Vries et al. 2005; Monteiroet al. 2005). Optimizing glucose and Mg2+concentration in asporulation medium led to a 17-fold increase in spore yield of aB. subtilis strain promoting plant growth (Posada-Uribe, Romero-Tabarez and Villegas-Escobar 2015). The mechanism by whichcell metabolism affects sporulation is complex. For instance,the sporulation process of B. cereusATCC14579 was substantiallylonger in presence of high glutamate concentration (de Vrieset al. 2005). However, different glutamate concentrations hadno effect on the temporal expression of sigF and sigG genesencoding the key transcriptional sporulation factors σ F and σG.Glutamate concentrationmay therefore affect the programmingof sporulation events that occur after those directly controlledby σG, which include mother cell lysis and spore maturation.

Factors influencing spore structure and compositionSpore structure and composition are also affected by changes inthe sporulation environment (Fig. 2). Spores accumulate min-erals in the spore core (mainly Ca2+, Mg2+ and Mn2+) duringthe sporulation process. Spore-coremineral concentrations varywidely and are highly dependent on the composition of thesporulation medium (Bassi, Cappa and Cocconcelli 2012). Thespore core is also characterized by a high content of CaDPA thatmay form, according to a recently proposed model, an inorganicpolymer bridged by water molecules that maintains the sporecore in an as-yet undetermined state described as glassy or gel-like (Setlow and Li 2015). Thiswater-CaDPA polymer favors sporeresistance by immobilizing proteins or molecules such as mem-brane lipids (Cowan et al. 2003; Cowan et al. 2004). Bacillus subtilisspores lacking the ability to synthesize DPA and that are formedin a growth medium without added DPA have a much highercore water content than spores formed in a DPA-supplementedmedium (Paidhungat et al. 2000). Spores of several Bacillus havea lower water content when formed on nutrient agar with a mixof metal cations (Ca2+, Mg2+, Fe2+, K+ andMn2+) than with Mn2+

only or without cations (Cazemier, Wagenaars and ter Steeg2001; Nguyen Thi Minh et al. 2011). Sporulation temperature isalso among the factors that have the strongest effect on DPA andcore water content, although this effect may not be systemati-cally reported (Melly et al. 2002; Kaieda et al. 2013). DPA concen-trations were higher in B. cereus and B. anthracis spores formedat high temperatures (30◦C and 45◦C, respectively) rather thanat low temperatures (10◦C and 25◦C, respectively) (Baweja et al.2008; Planchon et al. 2011). Water content was lower in B. sub-tilis spores formed at high temperature than at low temperature(Beaman and Gerhardt 1986; Melly et al. 2002; Nguyen Thi Minhet al. 2011). Moreover higher sporulation temperature has alsobeen correlated with higher levels of core spore mineralization(Palop, Sala and Condon 1999; Igura et al. 2003). The cortex pep-tidoglycan of spores prepared at different temperatures showedsubtle changes in structure, with slightly increased percentagesof cross-linked muramic acid in spores prepared at high tem-peratures (Melly et al. 2002). Cortex peptidoglycan structure wasalso substantially modified in spores of various Bacillus speciesproduced in a nutrient-poor mediumwithout any carbon source

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 617

DPA H2O

M+

Medium(with metal cations)

Medium(without C source)

Temperature Medium(agar vs broth)

Coat P

pH

Ger

Figure 2. A synthetic view on the major factors known to influence structure and composition of Bacillus spores. indicates an effect on a spore component orstructure. Effect on a spore component or structure with consequences on resistance to heat is indicated by , with consequences on resistance to chemical biocidesby , and with consequences on spore germination by . Coat P, coat proteins. Ger, germinant receptors. DPA, dipicolinic acid. M+, metal ions. H2O, spore corewater content. Ex, exosporium. Is, interspace. Ct, coat. Cx, cortex. Im, Om, inner and outer membrane. Co, core.

compared to a rich medium (Atrih and Foster 2001). The au-thors hypothesized that a change in muropeptide ratio, as wellas a probable decrease in number of muropeptides containingδ-lactam, reveals a functional defect of the cortex biosynthe-sis pathway and/or maturation machinery. Likewise, addition ofMnCl2 to sporulation media resulted in an altered peptidogly-can composition and peptidoglycan chain cross-linking. Mn2+

may therefore affect the expression of genes and/or the activ-ity of enzymes involved in cortex biosynthesis (Atrih and Foster2001).

Variations in sporulation temperature, pH or concentrationof inorganic salts resulted in substantial variations in spore vol-umes, which ranged from 0.38 μm3 to 0.79 μm3 for B. cereus and0.53 μm3 to 0.71 μm3 for B. megaterium (Zhou et al. 2017). Bacil-lus subtilis spores were nearly twice as small when formed ina Ca2+-deprived sporulation medium (Nguyen Thi Minh et al.2011). Spores of several B. cereus strains produced in liquid me-dia were significantly smaller than the ones formed on agar orin biofilms (van der Voort and Abee 2013). The roughness of thespore surface can be affected by incubation temperature or by awof the sporulation medium (Nguyen Thi Minh et al. 2011). Sporeswelling (shrinking) in response to high (low) relative humidityand core (de)hydration of B. thuringiensis and B. subtilis sporescould suggest a link between spore (de)hydration and spore size(Westphal et al. 2003; Sunde et al. 2009). However, spore hydra-tion is not the only cause: varying sporulation conditions led tomarked differences in spore volumes but without any differencein spore wet density (Zhou et al. 2017). Other structural modi-fications are directly observable with electron microscopy. Ex-amples are that B. cereus exosporium is damaged and detachedwhen spores are formed at high temperature (Faille et al. 2007),or that the outer coat of B. subtilis spores is thicker in sporesformed in a chemically-defined broth than in a rich agarmedium(Abhyankar et al. 2016).

Differences in coat protein profiles were observed for B. sub-tilis spores prepared at various temperatures or in broth vs. agarplates (Melly et al. 2002; Rose et al. 2007; Abhyankar et al. 2016). Incontrast α/β-type SASP remained unaffected in the same sporu-lation conditions (Melly et al. 2002; Rose et al. 2007). The inten-sity of the electrophoretic bands of the coat proteins CotA, CotG,CotB and CotS was lower in extracts from spores prepared at22◦C and 30◦C than at 48◦C. Several proteins of coat and ex-osporium extracts from B. cereus differed in spores formed at20◦C or 37◦C (Bressuire-Isoard et al. 2016). Among these, the CotEprotein was proportionally greater in extracts from spores pro-duced at 20◦C than at 37◦C. These differences may result fromdifferences in the amount and/or extractability of the proteins.In B. subtilis for instance, protein extraction yielded a higheramount of total proteins from spores prepared on agar than fromspores prepared in broth (Rose et al. 2007). Nevertheless, rela-tive coat protein contents did not show anymajor difference be-tween spores formed in agar vs. in broth (Driks 1999; Rose et al.2007).

Bacillus cereus and B. subtilis spores produced at differentsporulation temperatures showed significant differences in fattyacid (FA) composition. Total amount of anteiso FA increased inB. cereus spores produced at low temperature (Planchon et al.2011). The anteiso-to-iso ratio and the amount of unsaturatedFA in B. subtilis spore inner membrane increased as tempera-ture decreased (Cortezzo and Setlow 2005). These changes cor-respond to those generally observed in Bacillus spp. cells dur-ing low-temperature adaptation (Diomande et al. 2015) and likelyreflect a need to maintain membrane fluidity. A similar higheranteiso-to-iso FA ratio in the inner membrane was observed forspores formed on plates vs. broth (Rose et al. 2007).

Spore structure, organization and composition are clearlyvery sensitive to changes in the physical and chemical sporu-lation environment.

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

618 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

Sporulation conditions affect spore resistance

Many spore structures are involved in spore resistanceResistance to extreme temperatures is a distinguishing propertybetween bacterial spores and vegetative cells, as wet-heat inac-tivation of spores requires a roughly 45◦C higher temperaturethan wet-heat inactivation of vegetative cells (Setlow 2014a,b;Checinska, Paszczynski and Burbank 2015). Spore killing by wetheat is mainly due to damages to core proteins and denatura-tion of enzymes involved in metabolism (Coleman et al. 2007;Setlow 2014a,b; Wells-Bennik et al. 2016). Resistance to wet heatinvolves DNA saturation with α/β-type SASP proteins, low corewater, high DPA and mineral content, likely reducing molecularmobility in the core and protecting proteins against thermal de-naturation and irreversible aggregation (Sunde et al. 2009; Setlow2014a,b). The spore cortex, through the level of peptidoglycancross-linking, could also be involved in maintaining the dehy-drated state of B. subtilis spore cores (Atrih and Foster 1999; Driks1999). However, how such modifications in cortex compositionactually maintain spore core dehydration remains unknown.Increase in resistance during spore maturation concomitantlywith cross-linking of the outer coat proteins has recently sug-gested a possible role of coat in resistance to wet heat (Sanchez-Salas et al. 2011; Abhyankar et al. 2015). Spores of at least severalBacillus sp. share differentmechanisms for resistance to dry heatandwet heat. Resistance to dry heat is related to DNA saturationby α/β-type SASP in the core and activation of systems to re-pair dry heat-induced DNA damage during spore outgrowth likethe 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 ar-ray of physical insults including desiccation, freeze–thaw cycles,UV and γ -radiation, high hydrostatic pressure and chemical in-sults involving a variety of toxic effects (Nicholson et al. 2000;Setlow 2014a,b; Checinska, Paszczynski and Burbank 2015). Re-sistance to UV involves two major factors—α/β-type SASP bind-ing to DNA, andDNA repair during spore outgrowth—plusminorfactors such as carotenoids in spores outer layers, and lowwaterand high DPA content in spore core (Setlow 2014a,b). Spores are10–50 timesmore resistant to UV radiation than vegetative cells,depending on the species studied (Nicholson et al. 2000). Strongacid treatments, organic solvents at high temperatures, and ox-idizing agents such as hydrogen peroxide all cause major dam-ages in the inner spore membrane, where oxidation of mem-brane proteins may result in rupture and cell death (Cortezzoet al. 2004; Cortezzo and Setlow 2005). Alkali treatments mainlyinactivate the lytic enzymes of the coat that hydrolyze the cortexduring germination (Setlow et al. 2002). The spore coat is the firstline of defense against large molecules targeting the spore cor-tex, and it plays a major role in shielding the spore against oxi-dizing agents such as chlorine dioxide, hypochlorite or peroxyni-trite (Setlow 2000; Genest et al. 2002; Melly et al. 2002; Young andSetlow 2003). Resistance to chemicals involves a large numberof factors, such as detoxifying enzymes of spore coat and/or ex-osporium including catalase or superoxide dismutase, low per-meability of the spore inner membrane, DNA protection by α/β-type SASPs and delete DNA repair systems (Setlow 2006; Setlow2014a,b).

Factors influencing resistance to heatThe impact of environmental conditions on spore heat resis-tance was already observed back in the late 1920s on B. anthracisspores, which were more heat-resistant when formed at 37◦Cthan at 18◦C (Williams 1929). Spores of Bacillus sp. prepared at

Figure 3. 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, com-position of the sporulationmedium (carbohydrates andmineral compounds, pHand aw), method of spore preparation (agar, broth, biofilm) and of other fac-tors (ethanol, heat and pH shock, oxygen availability). In each considered pa-

per, the resistance to heat is expressed by decimal reduction time D, time to 2or 3 decimal reductions, or number of decimal reduction after a given time ofheat-treatment. A value of 1 ( ) has been arbitrarily attributed to the parameterexpressing 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), Mazaset al. (1997), Gonzalez et al. (1999), Movahedi and Waites (2000), Atrih and Fos-ter (2001), Cazemier, Wagenaars and ter Steeg (2001), Melly et al. (2002), Mova-

hedi 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), Mazaset al. (2009), Stecchini et al. (2009), Garcia, van der Voort and Abee (2010), Barilet 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).

suboptimal temperatures are consistently less resistant to wetheat than spores prepared at near-optimal temperatures (Bea-man and Gerhardt 1986; Condon, Bayarte and Sala 1992; Rasoet al. 1995; Gonzalez et al. 1999; Baweja et al. 2008; Garcia, van derVoort andAbee 2010; Baril et al. 2011; Nguyen ThiMinh et al. 2011;Planchon et al. 2011; Bressuire-Isoard et al. 2016). The differencecan be quite significant. Heat-resistance parameters such asdecimal reduction times, orD values, can vary by a factor greaterthan 10 depending on sporulation temperature (Fig. 3; Table S1,Supporting Information), and some authors have even proposedthe concept of an optimal sporulation temperature and pH atwhich spores get their maximal wet-heat resistance (Baril et al.2012; Trunet et al. 2015). Spore core water content decreases assporulation temperature increases and, as stated earlier under‘Factors influencing spore structure and composition’, there isa reciprocal positive correlation between core dehydration andspore wet heat resistance. DPA contributes to spore wet heat re-sistance by replacing water molecules and therefore maintain-ing low spore core water content. Similarly, a higher sporulationtemperature correlated with higher levels of spore mineraliza-tionwhich results in higher heat resistance (Palop, Sala andCon-don 1999). Sporulation temperature has subtle effects on the cor-tex structure of B. subtilis spores, with potential changes in core

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 619

hydration (Melly et al. 2002). Even a few minutes heat-shock orcold-shock at an appropriate time during sporulation can affectwet heat resistance. However, heat-shock and cold-shock pro-teins that usually accumulate in these conditions were not de-tectable soon after, and spores lacking different heat-shock pro-teins exhibited identical wet heat resistance towild-type spores,suggesting that they have no effect per se on spore resistance(Movahedi and Waites 2000; Melly and Setlow 2001; Movahediand Waites 2002). In contrast to heat resistance parameters,the zT-value, i.e. the temperature elevation causing a 10-foldreduction in D-values, remains relatively constant (from 8◦C to12◦C), as shown for instance for B. cereus and for B. licheniformisspores (Raso et al. 1995; Sala et al. 1995; Gonzalez et al. 1999). Boththe chemist energy of activation Ea and (food) microbiologist zTare expressing the effects of changes in temperatures on the in-activation reaction; zT may even be derived from Ea by a simplecalculation (Hoxey, Thomas and Davies 2007). This conserved zTvalue suggests that the basic mechanism of spore inactivationby wet heat remains constant whatever the sporulation temper-ature is, despite structural changes in spores and variable effi-ciency of spore heat killing.

Other growth conditions also have a significant impact onspore resistance to wet heat (Fig. 3; Supporting Information Ta-ble S1), but with no clear explanation and link with the sporestructure so far. Bacillus subtilis spores produced at low aw wereless resistant to wet heat than those formed at high aw (NguyenThi Minh, Perrier-Cornet and Gervais 2008). Bacillus anthracisspores formed at acid pH were found to be more resistant towet heat than spores formed at alkaline and neutral pH (Bawejaet al. 2008). The alkaline pH may increase mineralization ofspores during sporulation further contributing to heat resis-tance. Spores produced under low aeration or anaerobic condi-tions have a higher resistance to heat than spores produced un-der standard oxygen condition (Nguyen ThiMinh, Perrier-Cornetand Gervais 2008; Abbas et al. 2014). Mineralization of the sporu-lation media has a marked influence on the wet heat resistanceof spores, with a correlative effect on the water content of thespore core. Indeed the supplementation of sporulation media orthe remineralization with Ca2+of spores demineralized by acidtreatments generally tend to increase spore resistance to wetheat of species such as B. subtilis, B. megaterium, B. anthracis or B.licheniformis (Amaha and Ordal 1957; Cazemier, Wagenaars andter Steeg 2001; Igura et al. 2003; Baweja et al. 2008; Nguyen ThiMinh et al. 2011). However how mineralization affects the wa-ter content of the spore core is still poorly understood. Ca2+ en-richment within the CaDPA complex may limit the mobility ofwater molecules in the spore core (Setlow 2006). Bacillus cereusspores formed in a medium containing a high glutamate con-centration had a higher heat resistance than spores formed inpresence of low glutamate concentration (de Vries et al. 2005).While the chemical composition was not affected, resistance towet heat of B. cereus spores increased as sporulation mediumviscosity 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 dif-ferences in DPA, core water content and α/β-type SASPs, despitebeing significantly different in their wet heat resistance (Roseet al. 2007).

Sporulation conditions and resistance to physical and chemicalagents other than heatIn addition to wet heat resistance, the effect of sporulation con-ditions on resistance to a range of chemical and physical agentshas also been investigated. The properties of spores affected by

the sporulation conditions are very diverse, as the sporulationconditions influencing spore lead to modifications of properties(Table 1). However, for a given sporulation condition, the dif-ferent resistance properties are independently modified. For in-stance, increasing B. subtilis sporulation temperature from 22◦Cto 48◦C had a tremendous effect on wet heat resistance (D val-ues increased more than 10-fold) but no detectable effect ondry heat resistance and only a marginal effect on resistance toformaldehyde and some other DNA-damaging chemicals (Mellyet al. 2002; Cortezzo and Setlow 2005). This could be explained bysimilar concentrations of α/β-type SASPs, which are known to bemajor determinants of resistance to dry heat of spores formedat diverse temperatures (Melly et al. 2002).

The mechanisms underlying the observed effects of sporu-lation conditions on spore resistance to various chemical andphysical agents are generally not well understood, largely due tothe diversity of the tested sporulation conditions and inactiva-tion treatments and often to the absence of structural, biochem-ical ormolecular characterizations of spores. Supplementing thesporulation media with thioproline, cysteine or cystine resultedin significant increases in B. subtilis spore resistance to H2O2 andsolar UV radiation (290–400 nm) but not to 254-nm UV radiation(Moeller et al. 2011). This could be related to the potentially ra-dioprotective effects of these amino acids against reactive oxy-gen species generated by H2O2 and solar UV radiation. Moreover,de-coated spores lost this enhanced resistance, suggesting thatamino 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 becritical for sensitivity to DNA-damaging agents. Spores of B. sub-tilis formed in liquid media were more sensitive to nitrous acidand super-oxidized water than spores formed on solid media(Rose et al. 2007). Similarly, B. subtilis spores formed at lower tem-peratures were more sensitive to nitrous acid (Cortezzo and Set-low 2005) and they showed a higher rate ofmethylamine uptake,suggesting a more permeable spore inner membrane. Higher re-activity to deleterious chemicals in the spore core could also bedue to higher water content, as observed especially in sporesformed at low sporulation temperatures. However, the authorsnoted that even the significant differences in fatty acid profilesand core water contents were rather small and seem unlikely tobe the only cause of the observed effects (Cortezzo and Setlow2005). Other structuresmay be implicated, for instance the sporecoat, as spore coat elimination somehow increased the sensitiv-ity of spores to nitrous acid (Cortezzo and Setlow 2005).

Impact of sporulation environment on sporegermination

The sporulation environment also influences spore germinationand outgrowth after exposure to favorable conditions. Spore ger-mination is usually triggered by (i) nutrients, including sugars,purine nucleosides and amino acids, (ii) non-nutrient agentssuch as CaDPA, surfactants or dodecylamine or (iii) physicaltreatments such as hyperbaric treatment (Paidhungat et al. 2002;Setlow 2014a). Bacillus subtilis spores formed in a nutrient-poorsporulation medium germinated more slowly in response to L-valine and to a mixture of L-asparagine, D-glucose, D-fructoseand K+ (AGFK) than spores formed in a nutrient-rich medium(Ramirez-Peralta et al. 2012). This effect was likely due to a lowerlevel of the germinant receptor proteins and the lipoproteinGerD required for efficient B. subtilis germination with L-alanineobserved in the spores formed in the nutrient-poor medium

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

620 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

Table 1. Effects of sporulation conditions on the resistance of Bacillus sp. spores to physical and chemical agents other than wet heat.

Variable conditionsduring sporulation Species

Physical or chemicalagent Consequences on resistance Reference

Temperature B. subtilis Glutaraldehyde at 9 gL−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 chlorineAfter 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. After45 min logR ± 2 for spores produced at 48◦C.

Melly et al. (2002)

B. subtilis Sterilox at 240 mL L−1

free chlorineAfter 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 4min logR ± 1 for spores produced at 48◦C

Melly et al. (2002)

B. subtilis High pressure (300 Mpafor 60 min and at 55◦C)

logR = 2 for spores prepared at 30◦C; logR = 4 for sporesprepared at 37◦C or 44◦C

Igura et al. (2003)

B. subtils High osmolarity Osmoresistance with 2 mol L−1 NaCl was 4-fold higherfor spores formed at 25◦C than for spores formed at46◦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 andSetlow (2003)

B. subtilis Chloride dioxide at 2gL−1

After 10 min logR = 4 for spores produced at 25◦C andlogR±0 for spores produced at 46◦C.

Young andSetlow (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 andSetlow (2003)

B. subtilis Chloride dioxide at 2 gL−1

After 10 min logR = 4 for spores produced at 25◦C andlogR±0 for spores produced at 46◦C.

Young andSetlow (2003)

B. subtilis High pressure (800 MPaand 70◦C)

logR = 2.5 for spores prepared at 30◦C logR = 4 for sporesprepared 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 sporesformed at 47◦C

Deng et al. (2005)

B. weihen-stephanenis

NaOH 1M After 90 min logR = 0.5 for spores produced at 30◦C andlogR = 2.5 for for spores produced at 10◦C

Planchon et al.(2011)

B. weihen-stephanenis

Pulsed-UV light.Fluence = 0.7 J cm−2

logR = 2 for spores produced at 30◦C and logR = 4.5 forspores produced at 10◦C

Planchon et al.(2011)

Composition of thesporulation medium

B. subtilis UV in the range 280–400nm or 320–400 nm

Increasing F10b for spores prepared in mediasupplemented 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 andcomposition of thesporulation agar orbroth

B. sporother-modurans andB. amylolique-faciens (B.coagulans)

High pressure (500 MPa)and temperature (110◦C)

1.5–6 fold decrease in Dc-values when sporulationtemperature increased from 30◦C–37◦C (37◦C–50◦C).10-fold decrease to 2.5 increase in D withmineralization of the sporulation medium andsporulation temperature increase

Olivier, Bull andChapman (2012)

Anaerobiosis B. cereus 0.1 M Nitrous oxide After 120 min logR = 0.7 for spores produced inanaerobiosis 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, Bauerand Kulozik(2017)

pH, temperature B. subtilis High pressure at 350MPa 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 forspores formed at 19◦C

Nguyen Thi Minhet al. (2011)

Spore preparationmethod

B. subtilis Nitrous acid at 400mmol L−1 orsuper-oxidized water

Lag time in inactivation curves longer for sporesformed on agar plates longer than in broth

Rose et al. (2007)

alogR: number of log-reduction after a given treatment.bF10 : Fluence for a 10-fold reduction of the spore population.cLD90: or D, time to kill 90% of the spore population.

(Mongkolthanaruk, Robinson and Moir 2009; Ramirez-Peraltaet al. 2012). Bacillus cereus spores produced in a medium en-riched in amino acids and glucose showed enhanced germina-tion, together with increased levels of expression of the sevenger operons (Hornstra et al. 2006). Changes in expression of ger

operons caused variations in the number of germinant recep-tor proteins in the spore, and overexpression of gerA operons inB. subtilis also led to faster germination (Cabrera-Martinez et al.2003). Similarly, B. subtilis spores germinated more efficiently inresponse to moderate high pressure (150 MPa) when formed in

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 621

Table 2. Variations in germination of Bacillus spores according to sporulation conditions.

Variable conditionsduring 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 at20◦C

Baweja et al. (2008)

B. cereus High hydrostaticpressure

The ungerminated fraction of spores prepared at 20◦Cwas higher after HHP treatment in the range 250–700MPa than for spores prepared at 37◦C

Raso,Barbosa-Canovasand Swanson(1998a,b)

B. cereus Inosine and L-alanine Spores formed at 15◦C–20◦C are more sensitive andshow a higher germination rate than at 37◦C

Gounina-Allouane,Broussolle andCarlin (2008)

B. subtilis Dodecylamine > 90% germination within 50 min for spores preparedat 23◦C and < 25% for spores prepared at 44◦C

Cortezzo andSetlow (2005)

B. subtilis High hydrostaticpressure at 500 MPa

> 90% germination in 2.5 min for spores prepared at23◦C and 30◦C; <50% for spores prepared at 44◦C

Black et al. (2007)

B. subtilis L-valine 10 mM or 10mmol l−1 (each) AGFK

Rate of germination of spores prepared at 37◦C or 43◦Clower than of spores prepared at 23◦C or 30◦C

Luu et al. (2015)

B. weihen-stephanensis

12.5 mmol L−1 inosineand 25 mmol L−1

L-alanine

Faster germination with spores formed at 37◦C than at12◦C or 20◦C

Garcia, van derVoort and Abee(2010)

B. weihen-stephanenis

High hydrostaticpressure

Higher germination of spores formed at 20◦C and 37◦Cunder 150 MPa; no difference under 500 Mpa

Garcia, van derVoort and Abee(2010)

B. weihen-stephanenis

L-alanine at differentconcentrations

Spores formed at 10◦C are more sensitive to L-alanineand show a higher germination rate than at 30◦C

Planchon et al.(2011)

Composition of thesporulation agar orbroth

B. megaterium Diverse nutrientgerminants

Germination rate and spore activation requirementdepending on sporulation medium. Higher germinationrates for spores prepared with minerals and CaCl2supplementation

Levinson andHyatt (1964)

B. megaterium L-alanine or inosine No effect of heat-shock on germination of sporesformed on citrate-based medium. With acetate,heat-shock improves germination

Hitchins, Slepeckyand Greene (1972)

Composition of thesporulation agar orbroth

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 preparationmethod

B. subtilis Dodecylamine Slower germination of spores prepared on agar than onliquid

Setlow, Cowan andSetlow 2003; Roseet al. (2007)

Spore preparationmethod

B. cereus L-alanine 5 mmol L−1

and inosine 2.5 mmolL−1

Spores formed in biofilms showed a germinationcapacity lower than spores formed in broth

van der Voort andAbee (2013)

Spore preparationmethod

B. subtilis Moderate hydrostaticpressure at 150 MPa

Lower germination rate of spores formed in a poormedium

Doona et al. (2014)

NaCl concentration B. subtilis High hydrostaticpressure at 500 MPa

75% germination in 3 min for spores prepared withoutNaCl 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 MNaCl; Germination > control (No NaCl and CaCl2) forspores prepared with 1 M NaCl and 0.05 M CaCl2

Fleming and Ordal(1964)

Oxygen availability B. cereus L-alanine at differentconcentrations

> 90% (< 50%) germination for spores prepared inanaerobiosis (aerobiosis)

Abbas et al. (2014)

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 wasobserved in the spores formed in a nutrient-rich medium thanin a poor one. In contrast, the germination of both B. subtilis andB. weihenstephanensis spores at very high pressure (i.e.>500MPa),which is not germinant receptor-dependent, was not affected bysporulation temperature (Garcia, van der Voort and Abee 2010;Doona et al. 2014). Modulation of the quantity and/or activityof two exosporium enzymes, i.e. alanine racemase and the nu-cleoside hydrolase, may also affect nutrient-driven germination

(Todd et al. 2003; de Vries et al. 2005). Alanine racemase convertsL-alanine into D-alanine, which inhibits spore germination,while nucleotide hydrolase degrades inosine, amajor germinantfor spores of the B. cereus group.

Among other factors, sporulation temperature certainly hasamajor effect on spore germination (Table 2). However, high ger-mination rate or high germination efficiency has been associ-ated to either high or low sporulation temperatures dependingon strain, germinant, and conditions tested. In independent ex-periments for instance, sporulation temperature had oppositeeffects on the sensitivity of B. weihenstephanensis KBAB4 spores

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

622 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

to L-alanine alone or in combination with inosine (Table 2; Gar-cia, van der Voort and Abee 2010; Planchon et al. 2011).

Germination, mainly characterized by spore rehydration (re-vealed by the spore transformation from phase-bright to phase-dark) and loss of resistance, is only the very first stage in theprocess leading to the initiation of the first cell division and set-tlement of a daughter population. The question of whether laterpost-germination stages of spore evolution (duration of out-growth, or time to first cell division for instance) are affected bysporulation conditions has not been thoroughly studied. Modifi-cation of the sporulation environment triggers deep metabolicand physiological adaptations that cause structural modifica-tions in sporulating cells and alter spore properties. When re-lated to vegetative cells, including from Bacillus sp., these adap-tations create measurable effects on lag times during growth,and these effects have been extensively documented (Swinnenet al. 2004). However, this question has received less attentionin relation to spores. Sporulation at low aw, low temperature oralkaline pH favored colony formation of B. subtilis spores on a nu-trient agar at aw close to the limit of growth (Nguyen Thi Minhet al. 2011). However, absence of colony formation can signify im-paired germination or may equally signify impaired adaptationof germinated and/or outgrowing cells. With specific regard tooutgrowth capacity, B. weihenstephanensis spores formed at dif-ferent temperatures showed no significant change at a range ofincubation temperatures, despite sharp modifications of sporegermination and resistance to several stresses (Garcia, van derVoort and Abee 2010). Post-formation exposure of spores to non-lethal temperatures for several days resulted in different pat-terns of germination and outgrowth, associated to different lev-els of ribosomal RNA or proteins (Segev, Smith and Ben-Yehuda2012; Segev et al. 2013). There is no evidence that such differ-ences can be obtained by modifying sporulation conditions, butdifferences in the ‘molecular cargo’ gathered within spores cansignificantly affect revival. Is there really a spore ‘memory’, i.e.molecular or physical traces created during sporulation that willinfluence outgrowth and further growth events following germi-nation? This question receives an increasing attention. For in-stance, alanine-induced outgrowth of B. subtilis spores is depen-dent on the production during sporulation and on accumulationwithin spores of alanine dehydrogenase, a metabolic enzymethat converts alanine to pyruvate. More generally, this memorymay vary within the progeny of a sporulating cell population,and therefore could be beneficial to survive diverse selectionpressures in fluctuating environments (Mutlu et al. 2018).

CONCLUSIONS

Sporulation in the environment occurs in very diverse ecologi-cal niches (Carlin 2011; Gauvry et al. 2017). Bacterial spores willinexorably contaminate food-industry environments, health-care facilities and other microbiologically-sensitive sites, wheretheir resistance and recovery capacities will be largely un-known and practically unpredictable. Meanwhile, applicationsinvolving spore-forming bacteria are increasing and requirespores with reliable properties. Consequently, understandinghow sporulation in natural or industrial environments shapesspore properties is vital in order to design efficient microbialelimination or growth control strategies or, in contrast, to op-timize the requisite properties for applications in human andanimal health, crop protection, and other industrial domains. Inaddition, individual spores in populations show heterogeneousbehaviors in response to heat treatment or to suboptimal recov-

ery environments (Eijlander, Abee andKuipers 2011; Pandey et al.2013; van Melis et al. 2014; Warda et al. 2015). Whether and howspore properties can affect this variable pattern of behavior war-rants investigation in order to help better predict spores’ fate,and thus potentially improve important processes such as en-suring food safety and quality.

Laboratory studies show that sporulation media have astrong impact on spore properties, and that sporulation tem-perature is definitely one of the most important environmentalfactors influencing bacterial spore behavior. However, we can-not claim that lab studies provide a suitable description of theconditions occurring in the environment. Surveys on the ecol-ogy of spore-forming bacteria may contribute to help antici-pate and identify spatial and temporal distributions in termsof places and times when sporulation really occurs. In otherwords, does spore formation occur at low level in very differ-ent environments or as bursts at highly specific locations and/orduring or after specific and possibly rare meteorological events?This question was addressed in a review on spore-forming andthermophilicGeobacillus sp., but despitemultiple hypotheses, nofully satisfactory answer has emerged (Zeigler 2014).

Can we also infer the primary cause of differences in Bacil-lus spore resistance or germination from modifications in sporestructure and composition? Spore properties are usually clearlymodified by environmental conditions, but the link with struc-tural modifications or changes in spore composition is less thanclear and likely complex. Some processes are deeply multifac-torial, such as germination which is a cascade of molecular andbiochemical events.We cannot expect to identify sensitive sporebiomarkers to reliably predict spore properties and avoid costlyand time-consuming phenotypic characterization (of resistance,of germination and recovery, and so on) anytime soon. How-ever, we can anticipate greater support for the identification ofbiomarkers through the development of analytical tools. Ramanspectroscopy or fatty acid profile analysis, for instance, enableda fine discrimination of laboratory conditions (media) in whichB. cereus spores were formed (Ehrhardt et al. 2010; Dettman et al.2015). The omics techniques will also help decipher the complexnetwork of regulations (Bate, Bonneau and Eichenberger 2014;Abhyankar et al. 2017) that ultimately determines the desirableor undesirable properties of bacterial spores entering the worldof human activities.

SUPPLEMENTARY DATA

Supplementary data are available at FEMSRE online.

FUNDING

The PhD contract of CBIwas supported byMontpellier University(France) and Doctoral School GAIA.

Conflicts of interest. None declared.

REFERENCES

Abbas AA, Planchon S, Jobin M et al. Absence of oxygen affectsthe capacity to sporulate and the spore properties of Bacilluscereus. Food Microbiol 2014;42:122–31.

Abhyankar W, Pandey R, Ter Beek A et al. Reinforcement of Bacil-lus subtilis spores by cross-linking of outer coat proteins dur-ing maturation. Food Microbiol 2015;45:54–62.

Abhyankar WR, Kamphorst K, Swarge BN et al. The influ-ence of sporulation conditions on the spore coat protein

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 623

composition of Bacillus subtilis spores. Front Microbiol 2016;7:1–10.

Abhyankar W, Stelder S, de Koning L et al. ‘Omics’ for microbialfood stability: proteomics for the development of predictivemodels for bacterial spore stress survival and outgrowth. IntJ Food Microbiol 2017;240:11–18.

Amaha M, Ordal ZJ. Effect of divalent cations in the sporulationmedium on the thermal death rate of Bacillus coagulans varthermoacidurans. J Bacteriol 1957;74:596–604.

Atrih A, Foster SJ. The role of peptidoglycan structure and struc-tural dynamics during endospore dormancy and germina-tion. Antonie Van Leeuwenhoek 1999;75:299–307.

Atrih A, Foster SJ. Analysis of the role of bacterial endosporecortex structure in resistance properties and demonstra-tion of its conservation amongst species. J Appl Microbiol2001;91:364–72.

Avignone-Rossa C, Arcas J, Mignone C. Bacillus thuringiensisgrowth, sporulation and δ-endotoxin production in oxygenlimited and non-limited cultures. World J Microbiol Biotechnol1992;8:301–4.

Baril E, Coroller L, Postollec F et al. The wet-heat resistance ofBacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temper-ature but not on previous cell history. Int J Food Microbiol2011;146:57–62.

Baril E, Coroller L, Couvert O et al. Modeling heat resistance ofBacillus weihenstephanensis and Bacillus licheniformis spores asfunction of sporulation temperature and pH. Food Microbiol2012;30:29–36.

Barra-Carrasco J, Paredes-Sabja D. Clostridium difficile spores: amajor threat to the hospital environment. Future Microbiol2014;9:475–86.

Bassi D, Cappa F, Cocconcelli PS. Water and cations flux duringsporulation and germination. In: Abel-Santos E (ed). BacteriaSpores: Current Research and Applications. Norfolk, UK: CaisterAcademic Press, 2012, 143–67.

Bate AR, Bonneau R, Eichenberger P. Bacillus subtilis systems bi-ology: applications of -Omics techniques to the study of en-dospore formation. Microbiol Spectr 2014;2:1–15.

Baweja RB, Zaman MS, Mattoo AR et al. Properties of Bacillus an-thracis spores prepared under various environmental condi-tions. Arch Microbiol 2007;189:71–9.

Beaman TC, Gerhardt P. Heat resistance of bacterial sporescorrelated with protoplast dehydration, mineralization, andthermal adaptation. Appl Environ Microbiol 1986;52:1242–6.

Black EP, Wei J, Atluri S et al. Analysis of factors influencing therate of germination of spores of Bacillus subtilis by very highpressure. J Appl Microbiol 2007;102:65–76.

Boniolo FS, Rodrigues RC, Prata AMR et al. Oxygen supply inBacillus thuringiensis fermentations: bringing new insights ontheir impact on sporulation and delta-endotoxin production.Appl Microbiol Biotechnol 2012;94:625–36.

Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Micro-biol Rev 2010;23:382–98.

Bressuire-Isoard C, Bornard I, Henriques AO et al. Sporulationtemperature reveals a requirement for CotE in the assem-bly of both the coat and exosporium layers of Bacillus cereusspores. Appl Environ Microbiol 2016;82:232–43.

Cabrera-Martinez RM, Tovar-Rojo F, Vepachedu VR et al. Effectsof overexpression of nutrient receptors on germination ofspores of Bacillus subtilis. J Bacteriol 2003;185:2457–64.

Carlin F. Origin of bacterial spores contaminating foods. Food Mi-crobiol 2011;28:177–82.

Cazemier AE, Wagenaars SFM, ter Steeg PF. Effect of sporulationand recovery medium on the heat resistance and amount

of injury of spores from spoilage bacilli. J Appl Microbiol2001;90:761–70.

ChecinskaA, Paszczynski A, BurbankM. Bacillus and other spore-forming genera: variations in responses andmechanisms forsurvival. Annu Rev Food Sci Technol 2015;6:351–69.

Coleman WH, De C, Li YQ et al. How moist heat kills spores ofBacillus subtilis. J Bacteriol 2007;189:8458–66.

Condon S, Bayarte M, Sala FJ. Influence of the sporulation tem-perature upon the heat resistance of Bacillus subtilis. J ApplBacteriol 1992;73:251–6.

Cortezzo DE, Setlow P. Analysis of factors that influence the sen-sitivity of spores of Bacillus subtilis to DNA damaging chemi-cals. J Appl Microbiol 2005;98:606–17.

Cortezzo DE, Koziol-Dube K, Setlow B et al. Treatment with ox-idizing agents damages the inner membrane of spores ofBacillus subtilis and sensitizes spores to subsequent stress. JAppl Microbiol 2004;97:838–52.

Cowan AE, Koppel DE, Setlow B et al. A soluble protein is immo-bile in dormant spores of Bacillus subtilis but is mobile in ger-minated spores: implications for spore dormancy. Proc NatlAcad Sci USA 2003;100:4209–14.

Cowan AE, Olivastro EM, Koppel DE et al. Lipids in the innermembrane of dormant spores of Bacillus species are largelyimmobile. Proc Natl Acad Sci USA 2004;101:7733–8.

Cutting SM. Bacillus probiotics. Food Microbiol 2011;28:214–20.De Pieri LA, Ludlow IK. Relationship between Bacillus sphaericus

spore heat resistance and sporulation temperature. Lett ApplMicrobiol 1992;14:121–4.

de Rezende JR, Kjeldsen KU, Hubert CRJ et al. Dispersal of ther-mophilic Desulfotomaculum endospores into Baltic Sea sedi-ments over thousands of years. ISME J 2013;7:72–84.

de Vries YP, Atmadja RD, Hornstra LM et al. Influence of gluta-mate on growth, sporulation, and spore properties of Bacilluscereus ATCC 14579 in defined medium. Appl Environ Microbiol2005;71:3248–54.

Decker AR, Ramamurthi KS. Cell death pathway that monitorsspore morphogenesis. Trends Microbiol 2017;25:637–47.

Deng XT, Shi JJ, Shama G et al. Effects of microbial loading andsporulation temperature on atmospheric plasma inactiva-tion of Bacillus subtilis spores. Appl Phys Lett 2005;87:1539011–3.

Dettman JR, Goss JM, Ehrhardt CJ et al. Forensic differentiationof Bacillus cereus spores grown using different culture mediausing Raman spectroscopy.Anal Bioanal Chem 2015;407:4757–66.

Diomande SE, Nguyen-The C, Guinbretiere MH et al. Role of fattyacids in Bacillus environmental adaptation. Front Microbiol2015;6:1–20.

Doona CJ, Ghosh S, Feeherry FF et al. High pressure germi-nation of Bacillus subtilis spores with alterations in levelsand types of germination proteins. J Appl Microbiol 2014;117:711–20.

Driks A. Bacillus subtilis spore coat. Microbiol Mol Biol Rev1999;63:1–20.

Ehrhardt CJ, Chu V, Brown T et al. Use of fatty acid methylester profiles for discrimination of Bacillus cereus T-Strainspores grown on different media. Appl Environ Microbiol2010;76:1902–12.

Eijlander RT, Abee T, Kuipers OP. Bacterial spores in food:how phenotypic variability complicates prediction of sporeproperties and bacterial behavior. Curr Opin Biotechnol2011;22:180–6.

El-Bisi HM,Ordal ZJ. The effect of sporulation temperature on thethermal resistance of Bacillus coagulans var theromoacidurans.J Bacteriol 1956;71:10–16.

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

624 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

Elbisi HM, Ordal ZJ. The effect of certain sporulation condi-tions on the thermal death rate of Bacillus coagulans var ther-moacidurans. J Bacteriol 1956;71:1–9.

Eschlbeck E, Bauer SAW, Kulozik U. Effect of cultivation pH onthe surface hydrophobicity of Bacillus subtilis spores. AMB Ex-press 2017;7:1–7.

Faille C, Tauveron G, Gentil-Lelievre CL et al. Occurrence of Bacil-lus cereus spores with a damaged exosporium: consequenceson the spore adhesion on surfaces of food processing lines. JFood Prot 2007;70:2346–53.

Fleming HP, Ordal ZJ. Responses of Bacillus subtilis spores to ionicenvironments during sporulation and germination. J Bacteriol1964;88:1529–37.

Foster SJ, Popham DL. Structure and synthesis of cell wall, sporecortex, teichoic acids, S-layers, and capsules. In: SonensheinAL, Hoch JA, Losick R (eds). Bacillus subtilis and Its Closest Rel-atives: From Genes to Cells. Washington, DC: ASM Press, 2002,21–41.

Garcia D, van der Voort M, Abee T. Comparative analysis ofBacillus weihenstephanensis KBAB4 spores obtained at differ-ent temperatures. Int J Food Microbiol 2010;140:146–53.

Gauvry E, Mathot AG, Leguerinel I et al. Knowledge of the phys-iology of spore-forming bacteria can explain the origin ofspores in the food environment. Res Microbiol 2017;168:369–78.

Genest PC, Setlow B, Melly E et al. Killing of spores of Bacillussubtilis by peroxynitrite appears to be caused by membranedamage. Microbiology SGM 2002;148:307–14.

Giorno R, Bozue J, Cote C et al. Morphogenesis of the Bacillus an-thracis spore. J Bacteriol 2007;189:691–705.

Gonzalez I, Lopez M, Martınez S et al. Thermal inactivation ofBacillus cereus spores formed at different temperatures. Int JFood Microbiol 1999;51:81–84.

Gould GW. History of science–spores. J Appl Microbiol2006;101:507–13.

Gounina-Allouane R, Broussolle V, Carlin F. Influence of thesporulation temperature on the impact of the nutrients ino-sine and l-alanine on Bacillus cereus spore germination. FoodMicrobiol 2008;25:202–6.

Hayrapetyan H, Abee T, Nierop Groot M. Sporulation dynamicsand spore heat resistance in wet and dry biofilms of Bacilluscereus. Food Control 2016;60:493–9.

Henriques AO, Moran CP. Structure, assembly, and function ofthe spore surface layers. Annu Rev Microbiol 2007;61:555–88.

Hilbert DW, Piggot PJ. Compartmentalization of gene expressionduring Bacillus subtilis spore formation. Microbiol Mol Biol Rev2004;68:234–62.

Higgins D, Dworkin J. Recent progress in Bacillus subtilis sporula-tion. FEMS Microbiol Rev 2012;36:131–48.

Hitchins AD, Slepecky RA, Greene RA. Effect of carbon source onsize and associated properties of Bacillus megaterium spores.J Bacteriol 1972;110:392–401.

Hornstra LM, de Vries YP, de Vos WM et al. Influenceof sporulation medium composition on transcription ofger operons and the germination response of spores ofBacillus cereus ATCC 14579. Appl Environ Microbiol 2006;72:3746–9.

Hoxey EV, Thomas N, Davies DJG. Principles of sterilization. In:Denyer SP, Baird RM (eds). Guide to Microbiological Control inPharmaceuticals and Medical Devices. 2nd edn. Boca Raton, FL:CRC Press, 2007.

Igura N, Kamimura Y, IslamMS et al. Effects ofminerals on resis-tance of Bacillus subtilis spores to heat and hydrostatic pres-sure. Appl Environ Microbiol 2003;69:6307–10.

Imamura D, Kuwana R, Takamatsu H et al. Localization of pro-teins to different layers and regions of Bacillus subtilis sporecoats. J Bacteriol 2010;192:518–24.

Kaieda S, Setlow B, Setlow P et al.Mobility of corewater in Bacillussubtilis spores by 2H NMR. Biophys J 2013;105:2016–23.

Khoury PH, Lombardi SJ, Slepecky RA. Perturbation of the heatresistance of bacterial spores by sporulation temperatureand ethanol. Curr Microbiol 1987;15:15–19.

Knudsen SM, Cermak N, Delgado FF et al. Water and small-molecule permeation of dormant Bacillus subtilis spores. JBacteriol 2016;198:168–77.

Lechowich RV, Ordal ZJ. Influence of sporulation temperatureon heat resistance and chemical composition of bacterialspores. Can J Microbiol 1962;8:287–95.

Lee JK, Movahedi S, Harding SE et al. The effect of acid shock onsporulating Bacillus subtilis cells. J Appl Microbiol 2003;94:184–90.

Leggett MJ, McDonnell G, Denyer SP et al. Bacterial spore struc-tures and their protective role in biocide resistance. J ApplMicrobiol 2012;113:485–98.

Lequette Y, Garenaux E, Combrouse T et al. Domains of BclA,the major surface glycoprotein of the B. cereus exosporium:glycosylation patterns and role in spore surface properties.Biofouling 2011;27:751–61.

Levinson HS, Hyatt MT. Effect of sporulation medium on heatresistance, chemical composition and germination of Bacillusmegaterium spores. J Bacteriol 1964;87:876–86.

Lindsay JA, Barton LE, Leinart AS et al. The effect of sporulationtemperature on sporal characteristics of Bacillus subtilis A.Curr Microbiol 1990;21:75–9.

Logan NA. Bacillus and relatives in foodborne illness. J Appl Mi-crobiol 2012;112:417–29.

Lundgren L. Effect of variation of sporulation time and tempera-ture on thermostability of Bacillus cereus spores. Physiol Plant1967;20:392–9.

Luu S, Cruz-Mora J, Setlow B et al. The effects of heat activationon Bacillus spore germination, with nutrients or under highpressure, with or without various germination proteins. ApplEnviron Microbiol 2015;81:2927–38.

Maillard JY. Innate resistance to sporicides and potential failureto decontaminate. J Hosp Infect 2011;77:204–9.

Margosch D, GanzleMG, EhrmannMA et al. Pressure inactivationof Bacillus endospores. Appl Environ Microbiol 2004;70:7321–8.

Mazas M, Lopez M, Gonzalez I et al. Effects of sporulation pH onthe heat resistance and the sporulation of Bacillus cereus. LettAppl Microbiol 1997;25:331–4.

Mazas M, Fernandez A, Alvarez A et al. Effects of phosphate andsodium and potassium chlorides on sporulation and heat re-sistance of Bacillus cereus. J Food Saf 2009;29:106–17.

McKenney PT, Driks A, Eichenberger P. The Bacillus subtilis en-dospore: assembly and functions of the multilayered coat.Nat Rev Micro 2013;11:33–44.

McKenney PT, Driks A, Eskandarian HA et al. A distance-weighted interaction map reveals a previously uncharac-terized layer of the Bacillus subtilis spore coat. Curr Biol2010;20:934–8.

Melly E, Setlow P. Heat shock proteins do not influence wet heatresistance of Bacillus subtilis spores. J Bacteriol 2001;183:779–84.

Melly E, Genest PC, GilmoreME et al.Analysis of the properties ofspores of Bacillus subtilis prepared at different temperatures.J Appl Microbiol 2002;92:1105–15.

Moeller R, Wassmann M, Reitz G et al. Effect of radioprotec-tive agents in sporulation medium on Bacillus subtilis spore

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

Bressuire-Isoard et al. 625

resistance to hydrogen peroxide, wet heat and germicidaland environmentally relevant UV radiation. J Appl Microbiol2011;110:1485–94.

Mongkolthanaruk W, Robinson C, Moir A. Localization of theGerD spore germination protein in the Bacillus subtilis spore.Microbiology 2009;155:1146–51.

Monteiro SM, Clemente JJ, Henriques AO et al. A procedure forhigh-yield spore production by Bacillus subtilis. Biotechnol Prog2005;21:1026–31.

Movahedi S, Waites W. A two-dimensional protein gel elec-trophoresis study of the heat stress response of Bacillus sub-tilis cells during sporulation. J Bacteriol 2000;182:4758–63.

Movahedi S, Waites W. Cold shock response in sporulating Bacil-lus subtilis and its effect on spore heat resistance. J Bacteriol2002;184:5275–81.

Mutlu A, Trauth S, ZiesackM et al. Phenotypicmemory in Bacillussubtilis links dormancy entry and exit by a spore quantity-quality tradeoff. Nat Commun 2018;9:69.

Nguyen Thi Minh H, Guyot, S, Perrier-Cornet J-M. Effect ofthe osmotic conditions during sporulation on the subse-quent resistance of bacterial spores. Appl Microbiol Biotechnol2008;80:107–14.

Nicholson WL. Using thermal inactivation kinetics to calculatethe probability of extreme spore longevity: implications forpaleomicrobiology and lithopanspermia. Orig Life Evol Biosph2003;33:621–31.

Nguyen Thi Minh H, Durand A, Loison P et al. Effect ofsporulation conditions on the resistance of Bacillus subtilisspores to heat and high pressure. Appl Microbiol Biotechnol2011;90:1409–17.

Nicholson WL, Munakata N, Horneck G et al. Resistance of Bacil-lus endospores to extreme terrestrial and extraterrestrial en-vironments. Microbiol Mol Biol Rev 2000;64:548–72.

Oliva C, Turnbough CL, Kearney JF. CD14-Mac-1 interactions inBacillus anthracis spore internalization by macrophages. ProcNatl Acad Sci USA 2009;106:13957–62.

Olivier SA, Bull MK, Chapman B. Bacillus spp. spores producedat lower temperatures are more resistant to high pres-sure thermal processes but mineralization does not pre-dict relative resistance. Innov Food Sci Emerg Technol 2012;16:96–101.

Paidhungat M, Setlow B, Driks A et al. Characterization of sporesof Bacillus subtilis which lack dipicolinic acid. J Bacteriol2000;182:5505–12.

Paidhungat M, Setlow B, Daniels WB et al.Mechanisms of induc-tion of germination of Bacillus subtilis spores by high pres-sure. Appl Environ Microbiol 2002;68:3172–5.

Palop A, Sala FJ, Condon S. Heat resistance of native and dem-ineralized spores of Bacillus subtilis sporulated at differenttemperatures. Appl Environ Microbiol 1999;65:1316–9.

Pandey R, Ter Beek A, Vischer NOE et al. Live cell imaging of ger-mination and outgrowth of individual Bacillus subtilis spores;the effect of heat stress quantitatively analyzed with Spore-Tracker. PLoS One 2013;8:1–10.

Paredes-Sabja D, Sarker MR. Germination response of spores ofthe pathogenic bacterium Clostridium perfringens and Clostrid-ium difficile to cultured human epithelial cells. Anaerobe2011;17:78–84.

Perez-Garcia A, Romero D, de Vicente A. Plant protectionand growth stimulation by microorganisms: biotechnologi-cal applications of Bacilli in agriculture. Curr Opin Biotechnol2011;22:187–93.

Planchon S, Dargaignaratz C, Levy C et al. Spores of Bacillus cereusstrain KBAB4 produced at 10◦C and 30◦C display variations intheir properties. Food Microbiol 2011;28:291–7.

Posada-Uribe LF, Romero-Tabarez M, Villegas-Escobar V. Effectof medium components and culture conditions in Bacillussubtilis EA-CB0575 spore production. Bioprocess Biosyst Eng2015;38:1879–88.

Ramirez-Peralta A, Zhang PF, Li YQ et al. Effects of sporula-tion conditions on the germination and germination pro-tein levels of Bacillus subtilis spores. Appl Environ Microbiol2012;78:2689–97.

Raso J, Barbosa-Canovas G, Swanson BG. Sporulation temper-ature affects initiation of germination and inactivation byhigh hydrostatic pressure of Bacillus cereus. J Appl Microbiol1998a;85:17–24.

Raso J, Gongora-Nieto MM, Barbosa Canovas GV et al. Influenceof several environmental factors on the initiation of germi-nation and inactivation of Bacillus cereus by high hydrostaticpressure. Int J Food Microbiol 1998b;44:125–32.

Raso J, Palop A, Bayarte M et al. Influence of sporulation tem-perature on the heat resistance of a strain of Bacillus licheni-formis (Spanish Type Culture Collection 4523). Food Microbiol1995;12:357–61.

Rosales-Mendoza S, Angulo C, Meza B. Food-grade organisms asvaccine biofactories and oral delivery vehicles. Trends Biotech-nol 2016;34:124–36.

Rose R, Setlow B, Monroe A et al. Comparison of the properties ofBacillus subtilis spores made in liquid or on agar plates. J ApplMicrobiol 2007;103:691–9.

Sala FJ, Ibarz P, Palop A et al. Sporulation temperature and heat-resistance of Bacillus subtilis at different pH values. J Food Prot1995;58:239–43.

Schaeffer P, Millet J, Aubert JP. Catabolic repression of bacterialsporulation. Proc Natl Acad Sci USA 1965;54:704–11.

Sanchez-Salas JL, Setlow B, Zhang PF et al.Maturation of releasedspores is necessary for acquisition of full spore heat resis-tance during Bacillus subtilis sporulation. Appl Environ Micro-biol 2011;77:6746–54.

Segev E, Smith Y, Ben-Yehuda S. RNA dynamics in aging bacte-rial spores. Cell 2012;148:139–49.

Segev E, Rosenberg A, Mamou G et al. Molecular kinetics of re-viving bacterial spores. J Bacteriol 2013;195:1875–82.

Setlow B, Cowan AE, Setlow P. Germination of spores of Bacillussubtilis with dodecylamine. J Appl Microbiol 2003;95:637–48.

Setlow B, Atluri S, Kitchel R et al. Role of dipicolinic acid in resis-tance and stability of spores of Bacillus subtilis with or with-out DNA-protective alpha/beta-type small acid-soluble pro-teins. J Bacteriol 2006;188:3740–7.

Setlow B, Loshon CA, Genest PC et al. Mechanisms of killingspores of Bacillus subtilis by acid, alkali and ethanol. J ApplMicrobiol 2002;92:362–75.

Setlow B, Parish S, Zhang P et al. Mechanism of killing of sporesof Bacillus anthracis in a high-temperature gas environment,and analysis of DNA damage generated by various decon-tamination treatments of spores of Bacillus anthracis, Bacillussubtilis and Bacillus thuringiensis. J Appl Microbiol 2014;116:805–14.

Setlow B, Setlow P. Role of DNA repair in Bacillus subtilis sporeresistance. J Bacteriol 1996;178:3486–95.

Setlow P. Resistance of bacterial spores. In: Storz G, Hengge Aro-nis R (eds). Bacterial Stress Responses. Washington, DC: Amer-ican Society of Microbiology, 2000, 217–30.

Setlow P. Spore germination. Curr Opin Microbiol 2003;6:550–6.Setlow P. Spores of Bacillus subtilis: their resistance to and

killing by radiation, heat and chemicals. J Appl Microbiol2006;101:514–25.

Setlow P. Germination of spores of Bacillus species: what weknow and do not know. J Bacteriol 2014a;196:1297–305.

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022

626 FEMS Microbiology Reviews, 2018, Vol. 42, No. 5

Setlow P. Spore resistance properties. Microbiol Spectr 2014b;2:1–14.

Setlow P, Li L. Photochemistry and photobiology of thespore photoproduct: a 50-year journey. Photochem Photobiol2015;91:1263–90.

Sonenshein AL. Control of sporulation initiation in Bacillus sub-tilis. Curr Opin Microbiol 2000;3:561–6.

Stecchini ML, Spaziani M, Del TorreM et al. Bacillus cereus cell andspore properties as influenced by the micro-structure of themedium. J Appl Microbiol 2009;106:1838–48.

Sunde EP, Setlow P, Hederstedt L et al.The physical state of waterin bacterial spores. Proc Natl Acad Sci USA 2009;106:19334–9.

Swinnen IAM, Bernaerts K, Dens EJJ et al. Predictive modellingof the microbial lag phase: a review. Int J Food Microbiol2004;94:137–59.

Stewart GC. The exosporium layer of bacterial spores: a connec-tion to the environment and the infected host. Microbiol MolBiol Rev 2015;79:437–57.

Tan IS, Ramamurthi KS. Spore formation in Bacillus subtilis. Env-iron Microbiol Rep 2014;6:212–25.

Todd SJ, Moir AJG, Johnson MJ et al. Genes of Bacillus cereus andBacillus anthracis encoding proteins of the exosporium. J Bac-teriol 2003;185:3373–8.

Tovar-Rojo F, Cabrera-Martinez RM, Setlow B et al. Studies on themechanism of the osmoresistance of spores of Bacillus sub-tilis. J Appl Microbiol 2003;95:167–79.

Trunet C, Mtimet N, Mathot AG et al. Modeling the recovery ofheat-treated Bacillus licheniformis Ad978 and Bacillus weihen-stephanensis KBAB4 spores at suboptimal temperature andpH using growth limits. Appl Environ Microbiol 2015;81:562–8.

van der Voort M, Abee T. Sporulation environment of emetictoxin-producing Bacillus cereus strains determines spore size,heat resistance and germination capacity. J Appl Microbiol2013;114:1201–10.

van Melis CCJ, den Besten HMW, Groot MNN et al. Quantifica-tion of the impact of single and multiple mild stresses onoutgrowth heterogeneity of Bacillus cereus spores. Int J FoodMicrobiol 2014;177:57–62.

Verma N, Singh NA, Kumar N et al. Screening of different mediafor sporulation of Bacillus megaterium. Int J Microbiol Res Rev2013;1:068–73.

WardaAK, den BestenHMW, ShaN et al. Influence of foodmatrixon outgrowth heterogeneity of heat damaged Bacillus cereusspores. Int J Food Microbiol 2015;201:27–34.

Warriner K, Waites WM. Enhanced sporulation in Bacillus sub-tilis grown on medium containing glucose: ribose. Lett ApplMicrobiol 1999;29:97–102.

Wells-Bennik MHJ, Eijlander RT, den Besten HMW et al. Bacterialspores in food: survival, emergence, and outgrowth. Ann RevFood Sci Technol 2016;7: 457–82.

Westphal AJ, Price PB, Leighton TJ et al. Kinetics of sizechanges of individual Bacillus thuringiensis spores in responseto changes in relative humidity. Proc Natl Acad Sci USA2003;100:3461–6.

Widderich N, Rodrigues CDA, Commichau FM et al. Salt-sensitivity of sigma(H) and Spo0A prevents sporulation ofBacillus subtilis at high osmolarity avoiding death during cel-lular differentiation. Mol Microbiol 2016;100:108–24.

Williams OB. The heat resistance of bacterial spores. J Infect Dis1929;44:421–65.

Wolken WAM, Tramper J, van der Werf MJ. What can spores dofor us? Trends Biotechnol 2003;21:338–45.

Xue QO, Gu CF, Rivera J et al. Entry of Bacillus anthracis spores intoepithelial cells is mediated by the spore surface protein BclA,integrin alpha 2 beta 1 and complement component C1q. CellMicrobiol 2011;13:620–34.

Young SB, Setlow P. Mechanisms of killing of Bacillus subtilisspores by hypochlorite and chlorine dioxide. J Appl Microbiol2003;95:54–67.

Zeigler DR. TheGeobacillus paradox: why is a thermophilic bacte-rial genus so prevalent on a mesophilic planet? Microbiology2014;160:1–11.

Zhou KX, Wisnivesky F, Wilson DI et al. Effects of culture con-ditions on the size, morphology and wet density of spores ofBacillus cereus 569 and Bacillus megaterium QM B1551. Lett ApplMicrobiol 2017;65:50–6.

Dow

nloaded from https://academ

ic.oup.com/fem

sre/article/42/5/614/4998855 by guest on 24 January 2022