International Dairy Journal 12 (2002) 217–223

Bacterial endospores the ultimate survivors Abdelmadjid Atrih, Simon J. Foster* Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK Received 19 April 2001; received in revised form 3 October 2001; accepted 9 October 2001

Abstract Bacterial endospores are the most resistant living structures, known. Their high degree of resistance to many treatments (including heat and UV) is due to many factors and is governed by the unique spore structure. Spore core dehydration is a primary determinant of heat resistance. A specialised cell wall peptidoglycan layer, termed the cortex maintains dehydration. During germination the spore cortex is hydrolysed to allow outgrowth of the new vegetative cell. The germination-specific lytic enzymes (GSLEs) responsible for cortex hydrolysis have recently begun to be identified. Their position, in the dormant spore, outside the protection of the dehydrated core requires a special mechanism of heat resistance, which is so far unknown. Recent evidence has begun to elucidate the molecular basis not only for resistance but also how it is acquired, maintained and lost during germination. Understanding of resistance mechanisms may allow the design of novel sporicidal treatments. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Spores; Peptidoglycan structure; Spore mineral content; Spore properties

1. Introduction Under unfavourable conditions (nutrient deprivation), vegetative cells of the bacilli and clostridia are able to undergo a differentiation process named sporulation. This complex phenomenon leads to the formation of dormant endospores, which are able to survive thousands if not millions of years (Cano & Borucki, 1995). Bacterial spores have no metabolism and can withstand a wide range of environmental assaults including heat, UV and solvents. Dormant spores can be found in virtually every type of environment on earth. Despite such extreme dormancy, spores maintain an alert sensory mechanism, which enables them to respond to specific nutrients. This leads to germination and outgrowth to form a new vegetative cell.

2. Why spores are important for the food industry? As a result of their resistance properties, bacterial spores are very difficult to eliminate from the food environment. Thus food preservation techniques are *Corresponding author. Tel.: +44-114-222-4411; fax: +44-114-2728697. E-mail address: s.foster@sheffield.ac.uk (S.J. Foster).

aimed at reducing the numbers of bacterial spores to a level which is commercially acceptable (Brown, 1994). In foods, the spore in its dormant state is not hazardous. However, spore germination, outgrowth and proliferation leads to formation of vegetative cells which are responsible for spoilage and/or food poisoning (Ciarciaglini et al., 2000). Bacillus cereus and Clostridium botulinum are the most common spore forming bacteria incriminated in such hazards. B. cereus is one of the most important causes of foodborne disease in some countries (Aas, Gondrosen, & Langeland, 1992). Almost all toxins of B. cereus are produced during vegetative growth and secreted by the cells (Granum, 1994). B. cereus is the cause of two different types of food poisoning, the emetic and the diarrhoea type. The emetic type is caused by a nonprotein heat stable component (Turnbull, 1986; Krammer & Gilbert, 1989; Shinagawa, Otake, Matsusaka, & Sugii, 1992). However, the diarrhoea type food poisoning is caused by an enterotoxin consisting of one polypeptide chain (Shinagawa, Ueno, Konuma, Matsusaka, & Sugii, 1991; Granum & Nissen, 1993). The fact that psychrotrophic strains growing in pasteurised milk products are able to produce toxins makes this intoxication more widespread and underestimated in private homes (Granum, 1994). The number of botulism cases and the mortality rate have decreased in recent years. However, botulism

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remains a significant public health hazard (McClure, Cole, & Smelt, 1994). Many cases of botulism are associated with consumption of home-canned foods, because temperatures achievable by boiling are not sufficiently high to kill C. botulinum spores. The increased consumer demand for healthier and less processed foods have resulted in the reduction of chemical preservatives (NaNO2, level of sugar, etc) and development of new preservation techniques. Reduced levels of preservatives and alternative means of packaging (vacuum packaging) may affect stability of products with respect to growth and toxin production by C. botulinum. The fact that some strains of nonproteolytic C. botulinum of type B, E and F can germinate and multiply at temperatures as low as 3.31C, explains the requirement of ensuring that vacuum packaged food processing, composition and storage would protect against survival and growth of C. botulinum (Lund & Peck, 1994).

3. Spore structure Basic spore structure is conserved among all the endospore formers. Spores are assembled in a highly complex and co-ordinated fashion. Fig. 1 shows an electron micrograph of a spore of B. subtilis. The spore coat is a multilayered structure encasing the spore and is composed of as many as 25, often highly cross-linked, polypeptide species (Driks, 1999). The coat plays a role in resistance to chemical and enzymatic assault acting primarily as a permeability barrier (Driks, 1999; McDonnell & Russell, 1999; Riesenman & Nicholson, 2000; Russell, 1990). A B. subtilis mutant completely

Glucosaminidase

GlcNAc MurNAc

δ δ -lactam

GlcNAc MurNAc GlcNAc MurNAc L-Ala

Fig. 1. Electron micrograph of a section through a B. subtilis spore.

lacking the spore coat layers is highly sensitive to lysozyme but shows normal heat resistance (Riesenman & Nicholson, 2000). Beneath the coat is a thick layer of peptidoglycan, which can account for almost 10% of the total spore dry weight. The peptidoglycan consists of two layers, the thin inner primordial cell wall and the outer cortex. The primordial cell wall represents only 2 to 5% of the total peptidoglycan of the spores (Atrih, . . Zollner, Allmaier, & Foster, 1996; Atrih, Zollner, Allmaier, Williamson, & Foster, 1998). It prevents the loss of cellular integrity after germination and serves as template for peptidoglycan biosynthesis during outgrowth. The cortex has a unique structure and characteristics that are broadly conserved in a number of spore forming bacteria (Atrih & Foster, 2001; Meador-Parton & Popham, 2000). These include the occurrence of d-lactam predominantly at every alternate muramic acid residue and the low cross-linking index which occurs at only 2.9% of muramic acid in B. subtilis spores (Fig. 2) (Atrih et al., 1996; Popham, Helin,

Lytic transglycosylase

GlcNAc MurNAc

δ

L-Ala

GlcNAc MurNac GlcNAc

MurNAc

δ

L-Ala

D-Glu

single L-alanine tetrapeptide

D-Glu

meso-A2pm

meso-A2pm D-Ala

D-Ala

D-Ala tetrapeptide tetrapeptide

δ GlcNAc MurNAc

meso-A2pm D-Glu L-Ala

GlcNAc MurNAc

GlcNAc

Fig. 2. Basic structure of spore cortex peptidoglycan. Arrows show the representative action of GSLEs (glucosaminidase and lytic transglycosylase) involved in cortex hydrolysis during germination. GlcNAc, N-acetylglucosamine; MurNac, N-acetylmuramic acid; L-Ala, L-alanine; D-Glu, Dglutamic acid; meso-A2pm, meso diaminopimelic acid; D-ala, D-alanine; d; d-lactam.

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Costello, & Setlow, 1996a; Atrih & Foster, 1999). The muramic d-lactam moiety is not required for heat resistance but is necessary for substrate recognition by germination specific lytic enzymes (GSLEs), which specifically hydrolyse the cortex during germination (Atrih et al., 1996, 1998; Atrih & Foster, 1999; Chen, Miyata, Makino, & Moriyama, 1997; Makino, Ito, Inoue, Miyata, & Moriyama, 1994; Foster & Johnstone, 1987). The cortex is involved in the maintenance but not the establishment of the heat-resistant, dormant state (Ellar, 1978; Atrih et al., 1996). The spore core (cytoplasm), contains all the necessary metabolic components of the cell as well as DNA. In the dormant spore the core is dehydrated which renders its contents heat resistant (Gerhardt & Marquis, 1989; Marquis, Sim, & Shin, 1994). The enzymes contained in the core resume their function during germination, upon rehydration. The spore core is highly mineralised, containing mainly Ca2+, Mn2+ and Mg2+ which occur in a chelate with the spore specific component, dipicolinic acid. Also within the core are a high concentration of small acid soluble proteins. These are associated with the spore DNA and are involved mainly in UV resistance (Setlow, 1994). The inner membrane from dormant spores has been shown to have a compressed polycrystalline structure and be more viscous than the vegetative cell membrane to which it reverts during germination (Elmes, Wilkins, & Fitz-James, 1983; Stewart, Eaton, Johnstone, Barrett, & Ellar, 1980a). Alteration of the inner membrane affects germination properties and interaction of specific germinants with the spore inner membrane causes a change in membrane fluidity (Skomurski, Racine, & Vary, 1983).

4. Basis of spore resistance Spores have evolved as a survival mechanism able to resist harsh conditions. They show increased resistance properties compared to their vegetative counterparts (Table 1). The molecular basis for heat resistance depends on several factors. Proteins and enzymes are considered the major targets for heat killing of spores (Belliveau, Beaman, Pankratz, & Gerhardt, 1992). In general, enzymes in extracts of spores are inactivated at temperatures much lower than those required to inactivate the same enzymes within intact spores (Warth, 1980). However, it is still unclear which enzymes and other proteins are the critical targets for killing. 4.1. Dehydration Spore core dehydration occurs during sporulation and results in 0.5–1 g of water per g dry weight compared to

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Table 1 Resistance properties of bacterial endospores of Bacillus subtilis. Resistances are expressed as the approximate fold increase over vegetative cells (adapted from Setlow, 1994) Treatment

Vegetative cells

Wild-type spores

ab SASP spores

H2O2 UV Heat

1 1 1

300 8 25,000

230 0.6 2400

Note: H2O2, survival after 2.5 min exposure with 10% v/v; UV, dose to kill 90% population (J m 2); Heat, time needed to kill >90% of population at 651C.

3–4 g in vegetative cells (Gerhardt & Marquis, 1989). Protoplast dehydration occurs during sporulation and correlates with acquisition of heat resistance by bacterial spores (Nakashio & Gerhardt, 1985). The low core water content reduces the amount of water associated with spore proteins in the core, thus stabilising them to thermal denaturation (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000). Achievement of a reduced spore core water content requires accumulation of minerals and the development of the cortex. However, it is still unclear how cortex controls core water content (Driks & Setlow, 1999). 4.2. Cortex structure It has long been known that intact cortex is required for heat resistance (Imae & Strominger, 1976), also levels of heat resistance have been linked to cortex size (Murrell & Warth, 1965). The conserved structural features of spore peptidoglycan in many spore-forming bacteria are probably crucial for spore dormancy and heat resistance (Atrih & Foster, 2001; Atrih & Foster, 1999). The low spore peptidoglycan cross-linking index is certainly important for establishing a defined architecture of this polymer (Atrih & Foster, 1999). Recent work on biosynthesis of B. subtilis spore peptidoglycan has shown an apparent gradient of cross-linking across its span. However, this gradient is not necessary for core dehydration (Meador-Parton & Popham, 2000). It is suggested that the low cross-linking of spore peptidoglycan may be a balancing act between achieving the wall stability necessary for maintaining spore core dehydration, the wall flexibility/degradability required for outgrowth and attainment of spore core dehydration (Meador-Parton & Popham, 2000). Any modification of peptidoglycan architecture may have an effect on the stable conformation of GSLEs, which are most likely to be located in the cortex (Moriyama et al., 1999; Chen, Fukuoka, & Makino, 2000). The GSLEs are responsible for the hydrolysis of cortex during germination and the release from dormancy. The stable conformation of GSLEs is probably required for their protection

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mechanism, which is important for both dormancy and heat resistance. 4.3. Mineralisation and dipicolinic acid Bacterial spores accumulate minerals, which contribute to spore stability and heat resistance (Bender & Marquis, 1985; Marquis & Bender, 1985; Slepecky & Foster, 1959). Ca2+, Mg2+ and Mn2+ are the most abundant elements and are mostly located in the core (Stewart, Somlyo, Somlyo, Shuman, Lindsay, & Murrell, 1980b). The amounts and identities of the major cations in spores can be varied, by alteration in the metal ion content of the sporulation medium (Slepecky & Foster, 1959; Atrih & Foster, 2001). Mn2+ has been shown to affect sporulation in B. megaterium, B. subtilis and B. fastidiosus (Aoki & Slepecky, 1973; Vasantha & Freese, 1979). Addition of Mn2+ to sporulation media results in an increase in spore yield, stability, attainment of correct cortex structure and also an increase in heat resistance (Atrih & Foster, 2001). Mn2+ is involved in carbohydrate metabolism (Vasantha & Freese, 1979) and phosphoglycerate phosphomutase was identified as a strictly Mn2+ requiring enzyme needed for optimal sporulation of B. subtilis, B. cereus and B. megaterium (Vasantha & Freese, 1979; Oh & Freese, 1976; Watabe & Freese, 1979). Mn2+ also has a role in the expression of genes and/or enzymatic activities involved in cortex biosynthesis (Atrih & Foster, 2001). As well as Mn2+ other minerals (Mg2+, Ca2+, etc) also have a role in spore metabolism and resistance (Atrih & Foster, 2001). Availability of minerals in an appropriate ratio is probably important for the attainment of optimal spore properties (Atrih & Foster, 2001; Slepecky & Foster, 1959). High salt concentration may also be important in generating a more homogeneous spore population (Slepecky & Foster, 1959). Increased core mineralisation is often associated with decreased core water content, and this may contribute to greater spore resistance to heat (Beaman & Gerhardt, 1986). Unlike spore core minerals, which play a clear role in spore resistance, the role of dipicolinic acid is less defined. However recent findings on B. subtilis have shown that mutations in the spoVFA or spoVFB genes which encode DPA synthetase (Paidhungat, Setlow, Driks, & Setlow, 2000), results in an increase in spore core water and decreased heat and H2O2 resistance (Nicholson et al., 2000; Paidhungat et al., 2000). 4.4. Other parameters Sporulation conditions such as medium and temperature affect spore properties (Slepecky & Foster, 1959; Atrih & Foster, 2001). Also spores of the same strain prepared at high temperatures are more resistant to heat

than those prepared at low temperatures (Palop, Sala, & Condon, 1999; Setlow, 1994; Warth, 1978; Atrih & Foster, 2001). Heat shock proteins, whose level increases as the sporulation temperature rises may be important in this phenomenon (Heredia, Garcia, Luevanos, Labbe, & Garcia-Alvarado, 1997; Setlow, 1994). Spores of thermophiles are much more heat resistant than are spores of mesophiles, which in turn are more resistant than spores of psychrophiles (Gerhardt & Marquis, 1989; Warth, 1978). This increase in heat resistance could be due to the decrease of core water content but also to the increased thermostability of the proteins of thermophiles. Higher temperatures may stabilize tertiary and quaternary conformations of vital macromolecules (Beaman & Gerhardt, 1986). Repair of damage to macromolecules is another factor involved in spore resistance. Repair of protein damage during spore germination and outgrowth may be important for spore survival after heat treatment (Russell, 1990; Nicholson et al., 2000). It is well known that DNA damage accumulated in the dormant spore will induce synthesis of repair proteins upon germination (Setlow & Setlow, 1996). At least one protein is uniquely present in spores which is dedicated to the repair during germination and outgrowth of the DNA damage caused by UV irradiation of spores (Nicholson et al., 2000).

5. Germination Germination is a series of successive and degradative events triggered by specific germinants, which leads to the loss of typical spore properties. The signalling process, which occurs when the nutrient germinant binds to the receptor complex and subsequently activates spore germination-specific cortex lytic enzyme, is not yet known. Any food treatment that effectively targets this germination mechanism has the potential to confer ambient stability on a manufactured food. Studies have shown that heat, combined with additional controlling factors (pH, organic acids, preservatives) affect Bacillus species spore viability (Oloyede & Scholefield, 1994), outgrowth (Banks, Morgan, & Stringer, 1988) and germination (Ciarciaglini et al., 2000). An understanding of germination physiology, will be valuable for improving existing and the development of new sporicidal treatments. It is now well established that cortex hydrolysis is essential for spore outgrowth (Atrih et al., 1998; Atrih & Foster, 1999; Popham, Helin, Costello, & Setlow, 1996b). The recent application of muropeptide analysis by RP-HPLC to determine cortex structure and its dynamics during germination has shown how the cortex is hydrolysed during germination and the role of the different GSLEs in vivo (Atrih et al., 1998; Atrih &

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Foster, unpublished results). In B. subtilis at least 3 enzyme activities are involved in germination. These are lytic transglycosylase, glucosaminidase and a nonhydrolytic, likely epimerase. Glucosaminidase and epimerase activities show optimal activities at pH 5 and they are not essential for the germination process in the presence of lytic transglycosylase activity (Atrih & Foster, unpublished results). The products of lytic transglycosylase activity are mostly found in the germination exudate as single units. The lytic transglycosylase is an exo-enzyme, the first such enzyme to be found in a Gram positive bacterium (Atrih et al., 1998; . Atrih, Bacher, Korner, Allmaier, & Foster, 1999; Atrih & Foster, 1999). The major GSLE of B. subtilis is SleB which is responsible for the lytic transglycosylase activity (Boland, Atrih, Chirakkal, Foster, & Moir, 2000). SleB is located just inside the spore coat layer in the dormant spore and exists in a mature form lacking a secretion signal (Moriyama et al., 1999). How SleB is activated during germination triggering is unknown but it is in an operon with the gene encoding YpeB, which is required for its activity (Boland et al., 2000).

6. Effect of sporicidal treatments on germination and outgrowth 6.1. Heat Addition of lysozyme to the culture medium of heat altered spores increases the recovery of non-proteolytic C. botulinum (Sebald & Ionesco, 1972; Hauschild & Hilsheimer, 1977) and C. perfringens (Duncan, Labbe! , & Reich, 1972; Labbe! & Chang, 1995). This requirement of lysozyme for germination, despite the presence of a medium that supplies the nutrients that allow germination, demonstrates that heat inactivation of GSLEs (which are necessary for cortex hydrolysis) may have occurred (Duncan et al., 1972; Labbe! & Chang, 1995). There is no increase in recovery of spores of B. subtilis in the presence of lysozyme after heat treatment. This implies that other components involved in germination/ outgrowth are affected (Atrih & Foster, unpublished). This demonstrates that although bacterial spores share a conserved peptidoglycan structure (Atrih & Foster, 1999, 2001), the GSLEs involved in its hydrolysis show a diversity concerning structure, localisation and the likely activation mechanism (Atrih & Foster, 1999; Foster & Johnstone, 1988; Moriyama et al., 1999). In fact in B. subtilis SleB, which has lytic transglycosylase activity, is a major spore resistance determinant, as spores lacking SleB are 100 fold less resistant than wild type to heat treatment (901C, 60 min; Atrih & Foster, unpublished).

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6.2. NaOH Exposure of spores to sublethal NaOH results in extraction of some coat proteins (Atrih & Foster, unpublished results; Gould, Stubbs, & King, 1970; Duncan et al., 1972) and faster germination than untreated spores (Duncan et al., 1972; Atrih & Foster, unpublished). This may be due to removal of a permeability barrier. Here again the lytic transglycosylase activity is resistant to NaOH whereas all the other GSLE activities are sensitive. SleB is a major NaOH resistance factor, as a SleB mutation results in spores with almost 30 fold more sensitivity (Atrih & Foster, unpublished). 6.3. Hydrogen peroxide Hydrogen peroxide is extensively used for sterilisation and disinfection. Hydrogen peroxide at high concentrations causes lysis of spores as indicated by microscopy (King & Gould, 1969; Shin et al., 1994). In contrast, at lower concentrations (o1% v/v), it also kills spores but causes neither germination-like changes nor lysis (King & Gould, 1969; Shin et al., 1994). The generally proposed lethal action occurs through radical formation. Enzymes within spores can be inactivated during lethal exposure to H2O2 and survival of spores is greater when plated onto rich medium compared with minimal medium (Palop, Rutherford, & Marquis, 1996). Possible targets for H2O2 killing of spores are enzymes, which are mainly in the spore protoplast (Palop et al., 1996). Previous studies have shown that enzymes, which play a key role in the biology of spores after germination, are particularly vulnerable (Palop et al., 1996, 1998). In contrast to NaOH and heat, H2O2 treatment results in inactivation of the germination apparatus with the concomitant destruction of lytic transglycosylase (SleB) activity in B. subtilis (Atrih & Foster, unpublished).

7. Outlook for the future Since the observation by Koch over 100 years ago that B. anthracis spores could survive boiling, research on spore resistance has continued. However, despite massive amounts of effort, the mechanisms which make spores so resistant have remained elusive. It appears that resistance is multifactorial, with many contributing parameters. Thus targeting one specific component may not be effective. Recent molecular evidence has begun to identify components involved in high levels of resistance. The major GSLE of B. subtilis, SleB, is an example of a component which must be highly protected in the dormant spore. SleB is present in the aqueous environment of the spore cortex and so must have very specific and exquisite properties to allow it to remain

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heat resistant in the dormant spore. By an understanding of resistance mechanisms it may be possible to specifically target them. It is surprising that even after many years of research, traditional spore control regimes are still the most effective. Rational design of novel sporicidal treatments will require the molecular basis of spore resistance properties to be elucidated.

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