Peter SetlowDepartment of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA
6.2
Introduction
Dormant spores of the genera Bacillales and Clostridiales are among the most resistant life forms known. Given that some of these organisms cause food spoilage and/or disease, there has long been interest in mechanisms of spore killing and resistance. A variety of agents will kill spores including UV and γ-radiation, high pressure and wet heat. However, for many applications, in particu-lar for thermosensitive materials, chemicals are the agent of choice for spore killing, with such chemicals commonly termed spori-cides. In general, spores are resistant to disinfectants used to kill growing bacteria and are much more resistant to sporicides than are growing cells [1–3]. Resistance to different chemicals is acquired at various times during spore formation, depending on when spore components that are important in resistance are synthesized or assembled. Resistance to sporicides and common disinfectants is lost during spore germination as dormant spores return to active growth. In general, completion of spore germination is required for full loss of resistance to many chemicals, although degradation of DNA-protective proteins early in outgrowth is essential for full loss of resistance to some genotoxic chemicals.
This section will focus on spore resistance to chemical disin-fectants and sporicides. Mechanisms of spore resistance to such chemicals will be discussed, as will mechanisms of spore killing by sporicides, focusing primarily on spores of Bacillus subtilis. However, there will also be a summary of what is known about mechanisms for chemical killing and resistance of spores of Clostridium species. Previous reviews on spore resistance to and killing by chemicals include one in the previous edition of this book, as well as more recent reviews [1–9].
Spore structure
Much work shows that spore chemical resistance is due to novel spore structures and components [1–3]. Consequently, it is useful to review spore structures and components and their key proper-ties. Almost all work on mechanisms of spore resistance and killing has been with spores of B. subtilis, and it is reasonable to question whether these results are applicable to spores of other species and genera. Spores of Bacillales and Clostridiales species share the same general novel structural features (Figure 6.2.1) and components and may therefore be of particular interest.
Russell, Hugo & Ayliffe’s: Principles and Practice of Disinfection, Preservation and Sterilization, Fifth Edition. Edited by Adam P. Fraise, Jean-Yves Maillard,
Introduction, 121Spore structure, 121Variables affecting spore chemical resistance, 123Mechanisms of spore killing by chemicals, 124Factors important in spore resistance to various chemicals, 125Conclusions, 128Acknowledgments, 128References, 128
Bacterial Sensitivity and Resistance to Microbicides6
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Cortex and germ cell wallUnder the spore’s outer membrane there are two layers of PG, the large cortex and the thinner germ cell wall [17]. The germ cell wall PG structure appears identical to that of growing cell wall PG, and while the cortex PG structure is similar to that of germ cell wall PG, it has modifications. One of these modifica-tions, muramic acid-δ-lactam, allows cortex PG to be selectively degraded by endogenous cortex lytic enzymes (CLEs) in the first minutes of spore germination. In contrast, germ cell wall PG lacks muramic acid-δ-lactam and is not degraded by CLEs, eventually becoming the cell wall of the outgrowing spore. Although the spore’s PG layers are essential for spore viability, there is no known direct role for PG in spore chemical resist-ance. It could, however, react with and detoxify chemicals. Cortex PG is involved in some fashion in the maintenance and possibly the establishment of the spore core’s low water content, which is important in some spore chemical resistance (dis-cussed further on in this Chapter).
Inner membraneBeneath the germ cell wall is the spore’s inner membrane which acts as a permeability barrier to small molecules, includ-ing methylamine and even water [2, 18–20]. This membrane’s low permeability plays a major role in spore resistance to chemicals that can kill spores by damaging essential compo-nents in the spore’s central core. The inner membrane has additional novel properties, for example: (i) fluorescent probes in this membrane are largely immobile; and (ii) the membrane appears to be significantly compressed, since the inner membrane-bounded volume increases 1.5–2-fold early in spore germination without adenosine triphosphate (ATP) synthesis [21]. However, the reasons for the novel properties of the spore’s inner membrane are not known; its phospholipid com-position is not unique and can be altered without major changes in spore properties [22].
ExosporiumStarting from the outside and working inwards, spores of some species (e.g. pathogens such as Bacillus cereus and Bacillus anthra-cis) contain an outermost exosporium composed of proteins and glycoproteins [3, 10, 11], although spores of many species, includ-ing B. subtilis lack an exosporium. The exosporium proteins are unique to spores and many are unique to the exosporium, although some exosporium proteins are orthologs of proteins in the next spore layer, the coat [10–13]. One role of the exosporium is as a permeability barrier against enzymes and antibodies. In addition, the exosporium of B. anthracis spores at least contains enzymes that can detoxify reactive chemicals, and these enzymes can play a role in spore resistance [10, 14].
CoatThe next spore layer is the coat, composed largely of proteins, with ≥70 spore-specific proteins identified in the B. subtilis spore coat [10, 11]. In spores of many species, the coat contains several layers and contains large amounts of insoluble protein, some of which is cross-linked. The coat plays a major role in the resistance of spores to enzymes that can degrade the more internal pepti-doglycan (PG) layers of spores, and thus protects them against predators [11, 15, 16]. Coat proteins comprise c. 50% of spore protein, and may act as reactive armor to detoxify chemicals before they can attack essential spore components further within the spore. Like the exosporium, the coat also contains enzymes that can detoxify reactive chemicals [11].
Outer membraneBeneath the coat is the spore’s outer membrane, derived from the plasma membrane of the mother cell that engulfs the developing spore during sporulation. While this membrane is essential during sporulation, it is probably not important in spore resist-ance, and may not be a significant permeability barrier in dormant spores [2].
Figure 6.2.1 Schematic structure of a spore of Bacillus or Clostridium species. Note that spores of some species lack an exosporium, the sizes of the different spore layers are not drawn to scale, and the relative sizes of these layers can vary with the species/strain as well as the sporulation conditions.
Coat
Exosporium
Cortex
Core
Outer membrane Inner membrane
Germ cellwall
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Chapter 6.2 Resistance of Bacterial Spores to Chemical Agents
proteins protects the DNA against many, but not all, genotoxic chemicals. In addition to α/β-type SASP, the core of spores of most Bacillales species (but not Clostridiales) contains high levels of a single other type of SASP, termed a γ-type SASP. This protein is made in parallel with the α/β-type SASPs although its amino acid sequence is very different; it plays no known role in spore resistance. All SASPs are degraded by a SASP-specific protease early in spore outgrowth, and the amino acids ultimately gener-ated are important for spore metabolism and protein synthesis at this time.
Variables affecting spore chemical resistance
A number of variables can affect the chemical resistance of spores, although in many cases the mechanisms behind these effects are unclear. Individual spores in populations of both Bacillus and Clostridium species also exhibit heterogeneity in their properties, including the timing of spore germination and wet heat resistance [32–36]. It seems reasonable to expect that individual spores in populations will also exhibit heterogeneity in chemical resistance, but this has not been studied.
Species/strainMany data indicate that spores of different species or different strains of the same species have different chemical resistance [1, 3, 6]. These differences will obviously influence the choice of spores appropriate as surrogates for assessment of the efficacy of chemical treatments, and it is unlikely that spores of one single species will be the ideal surrogate for different chemical treatments.
The reasons for species-specific differences in spore chemical resistance are not known. However, given the importance of the spore coat and the permeability of the spore’s inner membrane in B. subtilis spore resistance to chemicals, and the general con-servation of these structures in spores of different species, it seems likely that differences in these structures are involved in species-specific differences in spore chemical resistance.
Sporulation conditionsPrecise sporulation conditions can also affect the chemical resist-ance of spores; variables such as temperature, the particular sporulation medium, divalent metal ion concentrations in the sporulation medium, and solid versus liquid media all have effects on spore properties [1, 3, 4, 6, 37]. In most cases the mechanisms behind the effects of these variables on spore chemi-cal resistance are not known, although it is known that sporula-tion temperature affects core water content, while divalent metal ion concentrations in sporulation media can affect the levels of cations associated with DPA [3, 23, 38–40]. Whether these effects on core water content are the reason for the effects of sporulation temperature on spore chemical resistance, and how levels of DPA’s associated cations affect spore chemical resistance, are, however, unknown.
CoreThe final spore layer is the central core containing DNA, RNA and most enzymes. There are three novel features of the spore core. First, for spores suspended in water, the percentage of core wet weight as water is only 25–50% (depending on the species), much lower than the water content of a growing cell protoplast (c. 80%) [23]. Second, c. 25% of the dry weight of the core is a 1 : 1 complex of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA; Figure 6.2.2) with divalent cations, predominantly Ca2+ (Ca-DPA) [23]. The developing spore accumulates DPA from the mother cell compartment of the sporulating cell late in sporula-tion, and DPA accumulation is important in reducing spore core water content [24, 25]. DPA and its associated divalent cations are excreted in the first minutes of spore germination, allowing a significant rise in core water content [3].
The core’s low water content is very important in many spore-resistance properties, and is likely essential for spore dormancy as proteins ≥20 kDa in the core are immobile [26]. However, the precise roles that DPA and low core water content play in spore chemical resistance are not clear. To some degree this uncertainty is because the physical states of water and DPA in the spore core are not known. It has been suggested that core water is in a glass-like state [27–29]. However, this idea has not been universally accepted [30], and recent work using nuclear magnetic resonance (NMR) spectroscopy to assess spore core water structure indicates that core water is not in a glass-like state [19]. Presumably the high level of DPA and its associated divalent cations plays a sig-nificant role in core water properties, but exactly how is not known. In addition, the great majority, if not all, DPA in the core is generally present as Ca-DPA, and while the concentration of this compound in the core greatly exceeds its solubility, its precise physical state is not known.
Finally, spore DNA is saturated with a spore-specific group of proteins called small acid-soluble spore proteins (SASPs) of the α/β-type, named for the major B. subtilis proteins of this type [31]. One or more α/β-type SASPs are found in spores of all Bacillales and Clostridiales species, and the amino acid sequences of these novel proteins are conserved within and between species but exhibit no significant sequence homology to other proteins. The α/β-type SASPs are synthesized late in sporulation within the developing spore, and the saturation of spore DNA with these
Figure 6.2.2 Structure of dipicolinic acid. Note that at physiological pH the two carboxyl groups will be ionized, and in spores are undoubtedly coordinated to divalent metal ions, predominantly Ca2+.
N
COOHHOOC
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then slowly released, affecting subsequent viability studies in the chambers [41].
Problems associated with the recovery of chemically treated spores also go beyond concerns about residual chemical agent, since chemicals often generate spore damage that is only condi-tionally lethal [2, 6]. Thus the precise pretreatment of spores or recovery media used following chemical exposure can have dra-matic effects on spore recovery and thus profound effects on apparent spore chemical resistance. Examples include increased recovery of: (i) glutaraldehyde-treated spores by brief alkali pre-treatment [42]; (ii) iodine-treated spores by addition of lactate to the recovery medium [43]; and (iii) alkali-treated spores by pre-treatment with lysozyme [44]. A major concern with spore killing by chemicals is thus to ensure that the killed spores are truly dead, and either unable to germinate or only to germinate after long lag times. Use of artificial germinants such as lysozyme can often be helpful in ensuring that, while apparently killed, spores cannot still germinate; in other words, they are indeed dead.
Mechanisms of spore killing by chemicals
Chemicals kill spores by a number of mechanisms including DNA damage, damage to the spore’s inner membrane, inactivation of one or more spore germination proteins, and inactivation of key spore core enzymes [2]. It is not surprising that different chemi-cals kill spores by different mechanisms (Table 6.2.1), and a par-ticular chemical may kill spores by multiple mechanisms. Most work elucidating the mechanisms of spore killing by chemicals has used B. subtilis spores, while the use of mutants has facilitated mechanistic studies. It will be important to learn if mechanisms of chemical killing of spores of all Bacillales and Clostridiales species are the same as for B. subtilis spores.
Spore killing by DNA damageA number of genotoxic chemicals kill B. subtilis spores by DNA damage [2] (Table 6.2.1). The evidence for this is that: (i) survi-vors in spore preparations given a mild treatment by such chemi-cals have a high percentage of mutations; (ii) loss of DNA repair capacity, often due to the loss of the RecA protein, results in increased sensitivity to the chemical in question; (iii) there is DNA damage in spores killed by such chemicals; and (iv) DNA
Spore purityPurity of spore preparations can often have marked effects on chemical resistance as measured by standard tests. Presumably impurities can react with and detoxify chemical agents, leading to an apparent increased spore resistance to the chemical agent. Indeed, the addition of blood or serum to spores often increases their apparent chemical resistance, presumably by chemical reac-tions between impurities and the agent in question [1, 6]. Spore impurities may also include the enzyme catalase, which can reduce hydrogen peroxide concentrations. This is a potentially serious problem as levels of catalase often increase in sporulating cells. Consequently, when sporulating cells lyse, catalase may adsorb to the spore coat/exosporium, and in some cases may even be integral components of the spore’s outer layers [10, 11, 14]. In view of the concerns noted above, it is essential that spore prepa-rations used for the assessment of chemical resistance are free of contaminating material.
Spore purity can be assessed in several ways. For example, by microscopic examination to ensure there is minimal contamina-tion with sporulating or growing cells or visible debris. However, microscopic examination will miss material such as nucleic acids and cell wall polymers that are released when sporulating cells lyse and that may adsorb to spores. Also, repeated centrifugation of spore preparations with attention to the appearance of the spore pellet can often indicate that the spore pellet is homogene-ous and consists only of single spores, both indications of high spore purity. It should also be kept in mind that in applied uses, spores may be in biological fluids such as blood, fecal matter or soil, and these materials may reduce the sterilization efficacy of chemicals compared with tests with purified spores.
Spore storage and recovery conditionsThere are numerous reports that storage of purified spores can alter their chemical resistance [1, 3, 6]. Spores can be stored dry, in water at c. 4°C, in ethanol at low temperatures, or frozen in water. Unfortunately there are no data on the best storage method to ensure stable spore chemical resistance, nor knowledge of what may change during storage that can affect spore chemical resistance.
Another factor that can alter the degree of spore chemical resistance is the conditions used for assessment of spore viability [1, 4, 6]. This is not a major problem for spore resistance to radiation or heat, since simply cooling spores or turning off light sources immediately removes any toxic effect and therefore elim-inates further effects on spore recovery. In contrast, toxic chemi-cals must be inactivated to ensure that residual agents do not affect spore recovery by inactivating the germinated spores and growing cells generated when treated spores are plated on recov-ery media. A variety of methods can be used to neutralize chemi-cal agents, and it is crucial that the neutralization method allows full recovery of untreated spores. The effects of residual chemical agents can be extremely insidious; for example in one case hydrogen peroxide vapor used for spore treatment in Plexiglas sample chambers was adsorbed by the acrylic glass itself and was
Table 6.2.1 Mechanisms of spore killing by various chemicals (from [2]).a
Mechanism of spore killing Examples of chemicals that kill spores by this mechanism
Chapter 6.2 Resistance of Bacterial Spores to Chemical Agents
kill spores by this mechanism [2, 53–56]. The evidence for this mechanism of killing is given by the following:1. Treated spores do not lose small molecules, in particular DPA, but DPA is released from spores treated by incubation at lower temperatures than from untreated spores.2. Spores given a mild treatment with such chemicals exhibit lower viability on “stressful” media such as those with high salt or minimal nutrients compared with salt-free or rich media, while untreated spores have similar viability on both high salt and minimal media; this behavior is typical of cells that have suffered some type of membrane damage [57–59].3. Spores treated with these chemicals exhibit increased perme-ability to small molecules such as methylamine, and spores’ major permeability barrier to methylamine is likely the inner membrane [2, 18, 20, 53].4. Spores treated with these agents have no DNA damage and often germinate normally, but the killed spores lyse after germina-tion is initiated even if the spores are germinated artificially in a hypertonic medium.Unfortunately, while available evidence suggests that damage to the inner membrane is the mechanism whereby this group of agents kills spores, the nature of this damage is unknown (although it is not to unsaturated fatty acids) [53]. Interestingly, if this damage is minimal, spores may survive but are sensitized to a subsequent wet heat treatment that is not lethal for untreated spores [53].
Factors important in spore resistance to various chemicals
In addition to the multiple mechanisms of spore killing by chemi-cals, spore chemical resistance is also due to multiple factors, with the most important factor varying for different chemicals (Table 6.2.2). Factors important in spore chemical resistance include: (i) DNA protection by α/β-type SASPs; (ii) repair of DNA damage during spore outgrowth; (iii) decreased core water content; (iv) the low permeability of the spore’s inner membrane; (v) the spore coat; and (vi) enzymes associated with the spore’s outer layers. A major factor in resistance of growing bacteria to some chemicals is cytoplasmic detoxifying enzymes such as catalase, superoxide dismutase and alkylhydroperoxide reductase [6, 60, 61]. However, while such enzymes are present in the spore core, they play no role in spore resistance [62], presumably because such enzymes are inactive due to the core’s low water content. Mechanisms of spore resistance to specific chemicals are summarized below (conclusions based on work with B. subtilis spores unless noted otherwise).
DisinfectantsDisinfectants used to kill growing bacteria on surfaces, hands and instruments, including alcohols, detergents, phenols and chlo-rhexidine, are almost always inactive against spores, even though the growing cells of spore-formers are sensitive to such agents [1,
repair genes are often induced during outgrowth of spores treated with such chemicals. In contrast to the results noted above with chemicals that kill spores by DNA damage, spores treated by many other chemicals, including some that are genotoxic in growing cells such as hydrogen peroxide exhibit: (i) no DNA damage; (ii) no induction of DNA repair genes in spore out-growth; and (iii) no decrease in resistance when DNA repair capacity is lost.
Spore killing by inactivation of spore core enzymesRecent work has strongly suggested that it is through inactivation of one or more spore core enzymes that wet heat kills spores of Bacillus species [33, 45–47]. There are also data suggesting that some peroxides can inactivate spore core enzymes, with this inac-tivation accompanying spore killing [48, 49]. However, inactiva-tion of key core enzymes has not been causally connected with spore killing by peroxides or other chemicals.
Spore killing by preventing germinationOne obvious way to inactivate spores is to render them incapable of germinating, since germination-defective spores cannot return to life. Many spore germination proteins including CLEs are in the spore’s outer layers [3], and such proteins are presumably susceptible to exogenous chemicals. While germination protein inactivation in such a manner does take place, spores that are dead for this reason could come back to life at a later time via very slow germination or could be germinated by an exogenous lytic enzyme.
The evidence that spore killing is due to a spore germination defect is shown by the loss of spores’ ability to germinate parallels spore killing and by viable spores that can be recovered from “apparently dead” spores by artificial germination, often with exogenous lysozyme. For several chemicals, including glutaralde-hyde, ortho-phthalaldehyde and perhaps hydrogen peroxide, inac-tivation of germination proteins may play some role in spore killing, although recovery of spores treated with these chemicals by artificial germination is never close to 100% [2, 6, 42, 43, 50–52]. However, alkali treatment, at least under relatively mild (for spores!) conditions (1 m NaOH at 24°C for several hours) clearly kills spores by inactivation of CLEs [44]. Apparently alkali-killed B. subtilis spores: (i) go through early steps in spore germi-nation; (ii) do not hydrolyze cortex PG during germination; and (iii) can be completely recovered by treatment with low levels of lysozyme.
Spore killing by damage to the inner membraneThe inner membrane is an extremely strong permeability barrier in the spore, preventing loss of core small molecules, and allowing only slow passage of exogenous chemicals into the core [18, 19, 53]. Many chemicals have been suggested to kill spores by damag-ing this membrane, such that when spores germinate and the core expands, the inner membrane ruptures leading to spore death [2, 53]. Oxidizing agents including hypochlorite, chlorine dioxide, organic hydroperoxides, superoxidized water and ozone probably
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high-resolution structure of an α/β-type SASP–DNA complex indicates that α/β-type SASP binding does not shield amino groups of purines in DNA’s major groove, the predominant sites of DNA alkylation by EMS [64]. Consequently, α/β-type SASPs have no effect on this DNA modification in vitro or in spores [63]. However, spores are more resistant to EtO and EMS than are growing cells, and one factor in spore EMS resistance is the low permeability of the spore’s inner membrane [20]; presum-ably this is also true for EtO.
FormaldehydeSpores are killed by formaldehyde through DNA damage, although the DNA damage has not been identified, and DNA repair is important in spore’s formaldehyde resistance [65]. The α/β-type SASPs are important in protecting the spore DNA against formal-dehyde, as α−,β− spores are more formaldehyde-sensitive than wild-type spores. DPA and/or spore core water content are also important in formaldehyde resistance [17], as is the permeability of the spore’s inner membrane, as spores with increased inner membrane permeability have decreased formaldehyde resistance [20]. However, the coat does not play a major role in spore for-maldehyde resistance.
NitriteNitrite kills spores by DNA damage, although the precise DNA damage has not been identified [66]. α/β-Type SASP binding to DNA plays a major role in spore DNA protection against nitrite, as does DNA repair in spore outgrowth. Increased perme-ability of the inner membrane as well as increased core water content are also associated with decreased spore nitrite resistance [19, 38].
PeroxidesPeroxides including hydrogen peroxide and organic hydroperox-ides can have genotoxic effects in various cell types [5, 60, 61]. However, these compounds do not kill wild-type spores by DNA damage [67, 68]. The saturation of DNA with α/β-type SASP is the major factor in spore resistance to hydrogen peroxide, as peroxides kill α−,β− spores largely by DNA damage. The spore coat plays a minor role in spore resistance to hydrogen peroxide, and an increased core water content and loss of DPA are also associ-ated with decreased hydrogen peroxide resistance [17, 25]. Several studies have also found that UV radiation can act synergistically with hydrogen peroxide in spore killing [69, 70].
Ozone, peracetic acid, bleach and chlorine dioxideIn contrast to results with peroxides, the potentially genotoxic agent peracetic acid does not kill α−,β− spores by DNA damage and the mechanism of spore killing by this agent is unclear [67]. Ozone, bleach and chlorine dioxide, which also are potential genotoxic agents, also do not kill α−,β− spores by DNA damage, and the coat is important in spore protection against these chem-icals [54, 55]. This is also the case for several commercial spori-cide formulations including Decon and Oxone™ [71]. These
4–7]. Resistance to these disinfectants is acquired at defined times in sporulation, and is lost when spore germination is com-pleted. Precise mechanisms of spore resistance to disinfectants are unknown, but have been inferred from the timing of acquisition of disinfectant resistance during sporulation and seem likely to involve changes in the inner membrane, PG and coat. However, mutants that disrupt spore coat assembly have little to no effect on spore disinfectant resistance.
Genotoxic chemicalsSpores are killed by a variety of genotoxic chemicals, including ethylene oxide (EtO), formaldehyde, hydrogen peroxide and other peroxides, bleach and other halogen-releasing agents [2]. A variety of spore components and structures are important in the protection of spore DNA against potentially genotoxic agents, and the importance of different protective factors varies depend-ing on the chemical.
Ethylene oxideSpores are killed by EtO via damage to DNA, and DNA repair during outgrowth is an important factor in spore EtO resistance, as recA spores are significantly more EtO sensitive than are wild-type spores [63]. Surprisingly, saturation of spore DNA with α/β-type SASP is not involved in DNA protection against EtO. This was concluded from a study of effects of α/β-type SASP on alkylation of DNA either in spores or in vitro by ethyl methyl-sulfonate (EMS), a chemical that alkylates DNA at the same positions as EtO. Spores lacking c. 80% of their α/β-type SASP (termed α−,β− spores) were as EMS-resistant as were wild-type spores, and saturation of purified DNA with a purified α/β-type SASP did not affect DNA alkylation by EMS. Analysis of a
Table 6.2.2 Factors important in spore resistance to various chemicals (from [2, 60]).a
Type of chemical Protective factorb
Genotoxic chemicals
Low permeability of spore’s inner membrane; DNA saturation by α/β-type SASP; DNA repair during spore outgrowth; low core water content
Oxidizing agents Detoxifying enzymes in spore’s outer layers; spore coat protein; low permeability of spore’s inner membrane; DNA saturation by α/β-type SASP
Dialdehydes Spore coatsDisinfectants Spore coats, perhaps cortex and inner membrane structureAcids and alkali Not understoodSupercritical CO2 Not understoodPlasma Spore coat; DNA saturation by α/β-type SASP; not yet
thoroughly studied
SASP, small acid-soluble spore protein.a Information is for spores of Bacillus subtilis.b Not all protective factors are important in protecting against all chemicals of any particular type.
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Chapter 6.2 Resistance of Bacterial Spores to Chemical Agents
growing cells. The reason for the residual resistance of “coatless” spores to oxidizing agents is not clear, although it could be the low permeability of the spore’s inner membrane [20]. “Coatless” B. subtilis spores also retain a significant amount of coat protein that could contribute to the enhanced oxidizing agent resistance of these spores [79].
DialdehydesThe dialdehydes glutaraldehyde and ortho-phthalaldehyde are used in some settings for decontamination of medical instru-ments [4, 6, 50, 51]. These compounds appear to kill spores by inactivating some germination protein, but which protein and whether there are other mechanisms of spore killing by these agents are not clear [42, 50, 51]. Resistance to these chemicals is highly dependent on the spore coat, as chemically decoated spores or spores that lack much coat protein due to mutation of a major coat assembly protein are much more sensitive to these dialdehydes.
Acid and alkaliSpores are extremely resistant to acids and alkali. The α/β-type SASPs and DNA repair are not important in spore resistance to acid or alkali, and the coat has only a minimal role in spore resist-ance to these chemicals [44]. However, coat proteins can be extracted by alkali, in particular, so alkali treatment can sensitize spores to agents against which the spore coats are likely major resistance factors [11, 80]. There is also a recent report suggesting that silicate can be taken up by spores of several Bacillus species (but not B. subtilis), deposited on a spore coat layer, and contrib-ute to spore acid resistance [81].
Alkali can inactivate the CLEs of B. subtilis spores and leads to apparent spore death because the treated spores cannot complete germination [44]. In contrast, spore treatment with strong acid causes “acid popping” whereby spores in strong mineral acid actually seem to explode due to rupture of spore’s outer layers, with extrusion of spore DNA [44, 82]. However, the mechanism of this “acid popping” is unknown.
PlasmaLow-temperature plasma is gaining popularity for sterilization of heat-sensitive materials, in particular medical devices [83]. A variety of different types of plasma exist, and these can contain many different types of potentially sporicidal components, including UV photons and radical species. Some data indicate that the spore coat, DNA repair and α/β-type SASP can be impor-tant in protection against radical species in plasma [83, 84], but precise killing components of plasma other than UV photons have not been identified.
Supercritical carbon dioxideThere has been a moderate amount of work recently on the killing of spores of Bacillus species by supercritical CO2 [85–88]. However, there has been no study of the mechanisms of spore killing by or resistance to this agent, nor on the precise agent
chemicals, with the possible exception of peracetic acid, probably kill spores by damaging the spore’s inner membrane (see below).
Hydrogen peroxideAs noted above, hydrogen peroxide does not kill spores by DNA damage despite it being a genotoxic agent. However, it can enter the spore core, as α−,β− spores are killed via DNA damage by hydrogen peroxide [68]. This suggests that hydrogen peroxide could kill wild-type spores by damaging one or more proteins in the spore core, and inactivation of core enzymes by peroxides can accompany spore killing [48, 49]. However, it has not been shown that this enzyme inactivation is a cause of spore killing and not simply an event that takes place after spores receive other lethal damage. Hydrogen peroxide treatment also can have effects on both spore germination and outgrowth [52], but again it is not clear if these effects are causes of spore killing. As noted above, catalase in spores’ outer layers can assist in protection against hydrogen peroxide. Polycyclic terpenoids, termed sporulenes, associated with B. subtilis spores’ outer layers (and possibly the outer layers of other spores as well) can also provide protection against hydrogen peroxide [72]. Elevated levels of manganese, most likely in the spore core, also result in higher resistance of spores of Bacillus megaterium to hydrogen peroxide, but this is not the case for B. subtilis spores [73, 74].
Oxidizing agents other than hydrogen peroxideA variety of oxidizing agents are or have been proposed to be useful to kill spores in applied settings, including ozone, organic hydroperoxides, superoxidized water, bleach, chlorine dioxide, formulations containing free halogens, dimethyldioxirane, t-butyl hydroperoxide plus a tetra-amido macrocyclic ligand (TAML) activator [75, 76] and a number of commercial formulations [2, 53–56, 66, 77]. Invariably these compounds do not kill spores by DNA damage, but by damaging the spore’s inner membrane such that when spores germinate this membrane then ruptures causing spore death [2, 53–56, 66, 76, 77].
The major factor protecting spores against most oxidizing agents is the spore coat, as spores lacking most or their coat protein due to chemical removal or absence of one or more pro-teins essential for the assembly of major coat layers are generally much more sensitive to oxidizing agents. The spore coats provide less protection against hydrogen peroxide than against other oxi-dizing agents [78]. As noted above, DNA protection by α/β-type SASPs is a major factor protecting spores against hydrogen per-oxide and other peroxides. However, the α/β-type SASPs are gen-erally not important in spore resistance to other oxidizing agents [2, 53–56, 66, 76, 77]. Another factor in spore resistance to some oxidizing agents is the presence of detoxifying enzymes such as catalase and superoxide dismutase in spore outer layers, the latter enzyme in particular in the exosporium of B. anthracis spores [10, 11, 14].
While the coat is the major factor in spore resistance to most oxidizing agents, spores lacking the great majority of their coat protein are more resistant to most oxidizing agents than are
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on mechanisms of spore killing by and resistance to these new treatments.6. What are the mechanisms that prevent spore killing by disin-fectants such as alcohols, detergents, phenol and chlorhexidine? There is only minimal knowledge of mechanisms of spore resist-ance to such agents, despite their widespread use. Since most disinfectants can have severe effects on membranes, it is possible that a spore’s likely novel inner membrane structure is important in spore resistance to disinfectants, but this has not been studied.
Overall, while there is certainly more known about mecha-nisms of spore killing by and spore resistance to chemicals than there was when the previous edition of this book appeared, there is still much to be learned.
Acknowledgments
Work in the author’s lab has received generous support over the years in grants from the National Institutes of Health, GM19698, and the Army Research Office.
References
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actually killing the spores. Supercritical CO2 alone requires rela-tively high temperatures to effectively kill spores. However, small amounts of additives such as hydrogen peroxide in supercritical CO2 allow effective spore killing at moderate temperatures and pressures. In one study [85], the great majority of spores that had their viability reduced by 5-log following exposure to supercriti-cal CO2 plus hydrogen peroxide exhibited no drastic changes in spore structure or permeability; a killing mechanism was not determined.
Factors important in chemical resistance of spores of clostridium speciesWhile much is known about factors important in the chemical resistance of spores of Bacillus species, primarily B. subtilis spores, much less is known about factors important in the chemical resistance of Clostridium spores. However, the α/β-type SASPs are important in the resistance of C. perfringens spores to genotoxic chemicals, including nitrite, hydrogen peroxide and formalde-hyde [89–91]. Indeed, a C. perfringens α/β-type SASP restores much of the nitrite resistance to B. subtilis spores lacking their own major α/β-type SASP [92]. The core’s water content is also important in resistance of C. perfringens spores to nitrite, and DPA is important in spore resistance to HCl, formaldehyde and hydrogen peroxide [93, 94]. Much of the recent information on the chemical resistance of C. perfringens spores was obtained through the use of various molecularly engineered mutant strains. As this technology becomes more widespread, information on the mechanisms of the resistance of spores of other Clostridium species should be forthcoming.
Conclusions
Much is now known about mechanisms of bacterial spore killing by, and resistance to, chemicals used for sterilization, but major questions remain unanswered including the following:1. Are mechanisms of spore killing by and resistance to chemical sporicides similar for spores of different Bacillus species as well as for spores of Clostridium species? Most work to date has used B. subtilis spores with only minimal data for spores of other species.2. Why is the coat so important in spore resistance to a variety of chemicals? It is hypothesized that spore coat protein acts as reactive armor to detoxify chemicals, but is this hypothesis correct?3. What is the damage, most likely to the spore’s inner mem-brane, whereby most oxidizing agents kill spores? The spore’s inner membrane has a number of novel properties and may have an unusual structure, but the structure of this membrane is not known.4. How do chemicals such as glutaraldehyde and hydrogen per-oxide kill spores? Despite moderate amounts of work on this question and extensive use of such chemicals in sterilization appli-cations, the lethal target(s) of these chemicals remain unknown.5. How do newer sterilization technologies such as supercritical CO2 and plasma kill spores? There has been only minimal work
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Chapter 6.2 Resistance of Bacterial Spores to Chemical Agents
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