CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 99-119 Vol. 3, No. 2 0893-8512/90/020099-21$02.00/0 Copyright © 1990, American Society for Microbiology Bacterial Spores and Chemical Sporicidal Agents A. D. RUSSELL Welsh School of , University of Wales College of Cardiff, Cardiff, CFJ 3XF, Wales

INTRODUCTION...... 99 THE BACTERIAL SPORE ...... 99 SPOROSTATIC AND SPORICIDAL ACTIVITY ...... 100 Group A: Sporostatic Compounds ...... 101 and cresols...... 101 Organic and esters ...... 101 ...... 101 QACs ...... 101 Biguanides ...... 101 Organomercury compounds...... 101 Group B: Sporicidal Compounds ...... 102 Glutaraldehyde ...... 102 Formaldehyde ...... 102 Other aldehydes...... 103 -releasing agents ...... 103 and iodophors ...... 103 Peroxygens ...... 103 Ethylene oxide ...... 104 P-Propiolactone ...... 104 Other gases...... 105 RECOVERY AND REVIVAL OF INJURED SPORES ...... 105 SPOROGENESIS, SUSCEPTIBILITY, AND RESISTANCE ...... 106 Sporulation ...... 107 Germination...... 108 Outgrowth ...... 110 OVERCOMING SPORE RESISTANCE ...... 110 MECHANISMS OF SPORICIDAL ACTION...... 111 MEDICAL AND OTHER USES OF CHEMICAL SPORICIDES ...... 112 Sporicidal Agents ...... 112 Inhibitors of Germination and Outgrowth ...... 113 CONCLUSIONS ...... 113 LITERATURE CITED...... 114

INTRODUCTION learning more about the ways in which sporicides act or spores resist their action, and due attention will be paid to Bacterial spores are highly resistant to chemical and these aspects. Finally, the clinical uses of sporicidal agents physical agents (25, 88, 89, 91, 102, 139, 158, 166, 171-174, will be discussed. 178, 207, 208, 224, 225). Processes designed to achieve General aspects of disinfection and are to be sterilization of food, pharmaceutical, medical, and other found in references 73, 101, and 183. These include details, products have thus, of necessity, had to take this high level when relevant, of sporicidal activity. Spore resistance is of resistance into account. Spores are also of importance in described by Russell et al. (177) and Gardner and Peel (72). other contexts, notably, (i) as food-poisoning agents (Clos- In the United States, commercially available disinfectants tridium botulinum, C. perfringens, and Bacillus cereus), (ii) are regulated by the Environmental Protection Agency and as etiological agents (C. perfringens and C. tetani) in some must be used according to the directions specified on their infections, and (iii) as sources of , toxins, and labels. Workers elsewhere should be familiar with regula- . Add to these the complex and fascinating series tions pertaining to their own country. of events that take place during sporulation, germination, and outgrowth and the stage is set for a comprehensive study encompassing many scientific and medical disciplines, sev- THE BACTERIAL SPORE eral of which are outside the scope of the present paper. This paper will deal with chemical sporicidal agents of the The most important sporeformers are members of the type. Such chemicals are comparatively few in genera Bacillus and Clostridium. Certain other , number and their activity is often susceptible to environmen- e.g., Sporosarcinae, Desulfomaculum, and Sporolactobacil- tal conditions, at least some of which can be readily con- lus spp. (52), can also form spores, but will not be considered trolled. Other agents that are bactericidal and sporostatic but here. True endospores are also produced by thermophilic not usually sporicidal will also be considered when relevant. actinomycetes. Thermoactinomyces vulgaris spores are More effective sporicidal action will only be achieved by highly refractile, do not take up simple stains, have a typical 99 100 RUSSELL CLIN. MICROBIOL. REV.

TABLE 2. Agents with bactericidal, sporostatic, and sporicidal activity Bactericidal agents Bactericidal agents Comment that are sporostatic that are sporicidal Group A Phenols None in group A Even high concentra- Organic acids and tions for prolonged estersa periods at ambient QACs temp are not spori- Biguanides cidal; may be Organomercurials sporicidal at ele- Alcohols vated temperatures Group B Glutaraldehyde All in group B Low concentrations Formaldehyde are sporostatic; Iodine compounds usually much higher concentra- Chlorine compounds tionertons are needed CX Hydrogen peroxide for sporicidal effect FIG. 1. "Typical" bacterial spore. The exosporium is present in Peroxy acids some, but not all, types of spores. EXO, Exosporium; OSC, outer Ethylene oxide spore coat; ISC, inner spore coat; CX, cortex-; GCW, germ cell wall; 13-Propiolactone PM, plasma membrane. a For example, the [methyl, ethyl, propyl, and butyl esters of para-(4)-hydroxybenzoic ]. spore structure, contain dipicolinic acid, and are heat resis- tant (173). (cortical membrane, germ cell wall, primordial cell wall) of The structure of a so-called typical bacterial spore is the cortex develops into the cell wall of the emergent cell depicted in Fig. 1. It is clear that the spore is a complex when the cortex is degraded during germination. entity, being composed of several different layers, some of Two membranes, the inner and outer forespore mem- which are implicated in their greater resistance than vegeta- branes, surround the forespore during germination. The tive cells to chemical or physical processes. The molecular inner forespore membrane eventually becomes the cytoplas- structure of the bacterial spore is considered in detail by mic membrane of the germinating spore, whereas the outer Ellar (57) and Warth (233). The germ cell (protoplast or core) forespore membrane persists in the spore integuments. and germ cell wall are surrounded by the cortex, external to The spore coats make up a major portion of the spore which are the inner and denser outer spore coats. An (139), consisting mainly of protein with smaller amounts of exosporium is present in some spores, but may surround just complex carbohydrates and lipid and possibly large amounts one dense spore coat. of phosphorus. The outer spore coat contains the alkali- In terms of its macromolecular constituents (Table 1), the resistant protein fraction and is associated with the presence protoplast is the location of RNA, DNA, dipicolinic acid, ofdisulfide-rich bonds. The alkali-soluble fraction is found in and most of the calcium, potassium, manganese, and phos- the inner spore coats and consists predominantly of acidic phorus present in the spore. Also present is a substantial polypeptides which can be dissociated to their unit compo- amount of low-molecular-weight basic proteins which are nents by treatment with sodium dodecyl sulfate. rapidly degraded during germination (187). From this brief consideration of the structure and compo- The cortex consists largely of peptidoglycan, some 45 to sition of the bacterial spore, it is obvious that several sites 60% of the muramic acid residues not having either a peptide exist for attack by biocides and equally obvious that the or an N-acetyl substituent but instead forming an internal spore can possess barriers which limit biocide penetration. It amide, muramic lactam (233). Peptidoglycan is the site of is the purpose of this review not only to describe the activity, action of lysozyme and of nitrous acid. A dense inner layer properties, and uses of sporicidal agents but also to consider their , insofar as this is known, how resistance may be presented by the spore, and how this may TABLE 1. Chemical composition of bacterial spores be overcome.

Composition component Comment SPOROSTATIC AND SPORICIDAL ACTIVITY Outer spore Mainly protein Alkali resistant; removed coat by disulfide bond-re- Comparatively few antibacterial agents are actively spori- ducing agents cidal (101, 173, 180). Even quite powerful bactericides may Inner spore Mainly protein Alkali soluble be inhibitory to spore germination or outgrowth or both, i.e., coat sporostatic, rather than sporicidal. Examples include phe- Cortex Mainly peptidoglycan Differs from peptidogly- nols and cresols, quaternary ammonium compounds can of vegetative cell (QACs), biguanides such as , organic mercury wall compounds, and alcohols (group A in Table 2). Sporicidal Core Protein, DNA, RNA, Unique spore proteins activity may, however, be achieved at elevated tempera- DPA,a divalent cat- associated with DNA ions tures. It is clear from Table 3 that concentrations effecting sporostasis are usually very close to those that inhibit a DPA, Dipicolinic acid. vegetative cell growth. VOL. 3, 1990 SPORICIDAL AGENTS 101

TABLE 3. Comparison of bacteriostatic and Organic acids and esters. Organic acids such as benzoic sporostatic concentrations and sorbic acids and esters (parabens) of para-(4)-hydroxy- Antibacterial Bacteriostatic concn Sporostatic concn are widely used as preservatives (102, 126, 180, agent (%, wt/vol) (%, wt vol) 196, 232). They are bactericidal but not sporicidal. The 0.0004-0.0016 0.0005 parabens inhibit the growth and toxin production of C. Cetylpyridinium chlo- 0.0005 0.00025 botulinum (165) and act at the germination stage. Their ride activity is only slightly affected over the pH range 4 to 8, Chlorhexidine diace- 0.0001 0.0001 whereas organic acids are most active in the undissociated tate or gluconate form, at low pH values. Chlorocresol 0.02 0.02 Alcohols. is rapidly lethal to nonsporing bacteria Cresol 0.08 0.1 0.2 0.2 when the is used at appropriate concentrations, but Phenylmercuric nitrate 0.00001-0.0001 0.00002 has no sporicidal activity (171, 173). The addition of 1% or acetate sodium or potassium hydroxide, various acids, or 10% amyl- to 70% alcohol is claimed to enhance a m-cresol sporicidal Based on reference 173. activity (208). Initial sporicidal activity of an ethanol-hy- pochlorite mixture is high but decreases on storage (40). It is also apparent from Table 4 that even chemicals that Other alcohols, namely, methanol (methyl alcohol), pro- are considered to be sporicidal require much higher concen- pan-1-ol, propan-2-ol (, isopropanol), phen- trations for this effect than for bactericidal activity. Also, a ethyl alcohol (phenylethanol), and octanol, also lack spori- time factor must be considered since spores must invariably cidal activity (102, 180). It is of interest, however, to note be exposed for longer periods. A recent example of this is that fresh mixtures of methanol (15%) and have described by Power and Russell (156), who demonstrate that a low sporicidal activity and that this activity increases as 2% alkaline glutaraldehyde will sterilize an inoculum of ca. the mixture ages, in contrast to ethanol-hypochlorite or 108 CFU of Escherichia coli, Staphylococcus aureus, and propan-2-ol plus hypochlorite, when the reverse applies (40, vegetative cells of B. subtilis per ml within 10 min at 22C, 51). Furthermore, increasing the methanol concentration to whereas B. subtilis spores require several hours. 25%, and especially to 50%, produces a rapid initial spori- Agents that are actively sporicidal (group B in Table 2) cidal action which can be maintained for at least 8 h after include aldehydes, halogens, peroxygens, and gaseous or preparation (51). Alcohols have been used for the selective vapor-phase disinfectants. The properties of chemical com- isolation of sporeforming bacteria (122). pounds in both groups A and B are considered below. QACs. The QACs can be considered derivatives of ammo- nium salts (NH4X) in which the hydrogen atoms are Group A: Sporostatic Compounds replaced by alkyl groups (R1 to R4). The sum of the carbon atoms in the four R groups is >10, and at least one of the R Chemical compounds in group A are not sporicidal but groups must have a chain length in the range C8 to C18. inhibit germination or outgrowth at concentrations similar to As a group, the QACs are bactericidal in low concentra- bacteriostatic ones. tions to nonmycobacterial, nonsporeforming, gram-positive Phenols and cresols. Even at high concentrations, phenolic bacteria, are less active against gram-negative bacteria, and disinfectants are poorly sporicidal (102, 180), 2.5 and 5% are not sporicidal (41, 181). Low concentrations are, how- (wt/vol) having little effect on B. subtilis spores even after ever, sporostatic (Table 3), the QACs inhibiting outgrowth 100 h at 25 or 37TC. In contrast, concentrations as low as but not germination (172, 173). Activity is markedly reduced 0.2% (phenol), 0.08% (cresol), and 0.02% (chlorocresol) in the presence of organic matter and is greater at alkaline (wt/vol) are all effective inhibitors of germination (173). than at acid pH. Furthermore, sporicidal activity is greatly accelerated when is chlorhexi- a Biguanides. The most important biguanide phenolics are used at elevated temperatures (33). Such used as the acetate and process, utilizing 0.2% (wt/vol) chlorocresol at 98 to 100'C, dine, which is (diacetate) gluconate was for many years a pharmacopoeial process in the United salts. It is an effective bacteriostatic and bactericidal agent Kingdom for sterilizing certain injectable and ophthalmic towards many gram-positive and gram-negative bacteria, but products, but is no longer official. is not mycobactericidal and is sporostatic rather than spori- cidal (172, 173). Chlorhexidine is sporicidal at elevated temperatures (77, 190) and, like the QACs, it inhibits out- TABLE 4. Comparison of bactericidal and growth rather than germination (189). Activity is greatly sporicidal concentrations reduced in the presence of organic matter and is greater at alkaline than at acid Antibacterial Bactericidal concn Sporicidal concn pH. agent (%, wt/vol) (%, wt/vol) Organomercury compounds. In the last century, it was claimed that the inorganic mercury compound mercuric Chlorocresol 0.1 >0.4 chloride was rapidly sporicidal towards B. anthracis. It was Cresol 0.3 >0.5 subsequently demonstrated that this incorrect conclusion Phenol 0.5 >5.0 a failure to control in Phenylmercuric nitrate 0.002 >0.02 was based on adequately sporostasis Chlorhexidine diacetate 0.002 >0.05 the subculture medium (reviewed in reference 174). Organo- 0.002 >0.05b mercury compounds such as phenylmercuric nitrate, phe- Glutaraldehyde <0.1 2.0 nylmercuric acid, and thiomersal (merthiolate) are important Formaldehyde <1 4-8 preservatives in many types of pharmaceutical products. Hypochlorite 1-2 ppm 20 ppm These compounds are bacteriostatic and bactericidal and are at low but are a Kill may depend on pH and temperature and on period of treatment. effective sporostatic agents concentrations, b Not sporicidal at this concentration at ambient temperatures. only sporicidal when used at high temperatures (171, 173). 102 RUSSELL CLIN. MICROBIOL. REV.

Group B: Sporicidal Compounds based on acid rather than alkaline glutaraldehyde, thereby benefiting from the stability inherent in such solutions. The Chemical agents in group B are sporostatic at low concen- improved sporicidal activity claimed for these acidic solu- trations and sporicidal at much higher levels. tions has often been obtained by the addition of agents that Glutaraldehyde. Glutaraldehyde [pentanedial; CHO produce a potentiated or synergistic effect with the dialde- (CH2)3 CHO] is a powerful agent and elec- hyde, e.g., nonionic (28-31) and anionic surfac- tron microscope fixative (84, 185). Its activity depends on tants (81). Inorganic cation-anionic combinations pH, alkaline solutions being considerably more effective greatly increase the antimicrobial activity at acid pH with a than acid ones (84, 185, 186). There is, however, a complex further increase in efficiency at higher temperatures (81). relationship among the parameters of concentration, temper- These solutions have a shelf life of years in terms of ature, and pH. The rate of bactericidal and sporicidal activ- glutaraldehyde concentration, polymerization, and pH. ity for aqueous acid solution is considerably lower than that Babb et al. (11) have examined different glutaraldehyde for activated alkaline solution (26, 27, 84, 120, 137, 212). As formulations and showed that acid dialdehyde preparations, temperature is increased, however, this difference between although more stable than the alkaline ones, were less alkaline and acid solutions is reduced (84, 192, 213). sporicidal and more corrosive. One of the earliest indications of antimicrobial activity of An enhanced activity is claimed for a combination (Spori- glutaraldehyde arose from a survey of the sporicidal activity cidin; The Sporicidin Co., Washington, D.C.) of glutaralde- of saturated dialdehydes in a search for an efficient substitute hyde with sodium phenate and phenol (106, 107). Studies for formaldehyde. This search revealed (150) that glutaral- from our laboratory (E. G. M. Power and A. D. Russell, J. dehyde in alcoholic solution was superior as a sporicidal Appl. Bacteriol., in press) agree to claims that the undiluted agent to both formaldehyde and glyoxal. Stonehill et al. (206) mixture is sporicidal but not that a 1:16 dilution is sporicidal. and Snyder and Cheatle (194) demonstrated that aqueous Inadequate neutralization of the high phenate concentration solutions of the dialdehyde were acidic and needed to be may be a contributory factor in reaching an erroneous buffered ("activated") by suitable alkalinating agents to a conclusion. pH of 7.5 to 8.5 for antimicrobial activity. A 2% (wt/vol) Formaldehyde. Formaldehyde (methanal, HCHO) is used glutaraldehyde solution activated with 0.3% (wt/vol) sodium in both the gaseous and liquid forms (171-173). Formalde- bicarbonate was advocated to provide the minimum concen- hyde solution (Formalin) is an aqueous solution containing tration and conditions necessary for rapid sporicidal activity. ca. 34 to 38% (wt/wt) CH2O and methanol to delay polymer- At this in-use concentration, the dialdehyde was capable of ization. Formaldehyde is bactericidal and sporicidal, but at a killing spores of Bacillus and Clostridium spp. in 3 h (26, slower rate than glutaraldehyde (170). It combines readily 203). Rubbo et al. (170) reported a 4-log (99.9%) kill of spores with proteins and is less effective in the presence of organic of B. anthracis and C. tetani in 15 and 30 min, respectively. matter. Not all species are equally susceptible to glutaraldehyde, Ortenzio et al. (146) claimed that formaldehyde solution and B. subtilis (29) and B. pumilus (170) appear to be the was rapidly sporicidal to B. subtilis but not to C. sporogenes, most resistant. With B. subtilis spores in liquid suspension, which was not killed after 2 h of exposure. Borax-Formalin a 3-h contact period with 2% alkaline glutaraldehyde pro- and formaldehyde-alcohol have been found to destroy B. duces ca. a 6-log drop in viable count (73, 116, 131, 192). anthracis, C. perfringens, and C. tetani (199), although some Using the Association of Official Analytical Chemists spori- doubt must remain about the validity of these results since cidal test (8) and vacuum-dried spores of B. subtilis, how- there could have been a failure to neutralize formaldehyde in ever, Boucher (29) found that 10 h was necessary to achieve subculture media. A lack of sporicidal activity of 8% form- a complete kill. Obviously, this long period cannot always be aldehyde has been noted by Pepper and Chandler (150), and used in practice. The possible revival of glutaraldehyde- this finding might take on an added significance when linked treated spores will be considered later. to proposals that spores apparently inactivated by formalde- Vegetative bacteria are more susceptible to the action of hyde may be revived by appropriate posttreatment proce- glutaraldehyde, a concentration as low as 0.02% alkaline dures (200, 201). This aspect is considered in more detail aldehyde achieving an inactivation factor of 104to 106 within later. 20 min at 20°C (164). Irrespective of whether aqueous or alcoholic solutions of At alkaline pH, glutaraldehyde solutions have a tendency formaldehyde are used, time-survivor curves of treated to polymerize; polymerization in acid solution is very slow. bacterial spores often show an initial shoulder (170), al- Consequently, since monomeric glutaraldehyde is consid- though this is by no means a universal finding (219). Accord- ered to be the active moiety (170) with interaction between ing to some workers (170), various alcohols (methanol, amino groups in protein enhanced at alkaline pH (179), it is ethanol, and propan-2-ol) reduce the sporicidal activity of clear that acid solutions are more stable but less active and formaldehyde. The sporicidal activity of formaldehyde is alkaline solutions are less stable but more active. These influenced markedly by temperature, with extensive spore problems of stability and active life have prompted the inactivation at temperatures of 40°C and above (219). development of novel formulations to overcome these draw- Formaldehyde vapor may be obtained in various ways: (i) backs in use. Alkalination of glutaraldehyde produces a by evaporating appropriate dilutions of standardized batches gradual decrease in aldehyde concentration (25, 155), the fall of commercial Formalin (containing 10% methanol); (ii) by being temperature dependent (213). Some formulations uti- heating paraformaldehyde or the formaldehyde polymers, lize the benefits conferred by formulating in the lower urea formaldehyde and melamine formaldehyde, under con- alkaline range of ca. pH 7.5. One such product, a stabilized trolled conditions of time and temperature (221). Bacterial glutaraldehyde solution (131), also contains surfactants to spores and nonsporulating bacteria are fairly readily killed promote rinsing of surfaces and is claimed to have the usual by formaldehyde gas (207). A linear relationship exists antimicrobial activity while also maintaining a stable glutar- between the formaldehyde concentration and killing rate. aldehyde concentration and pH over 28 days. In many Nordgren (144) observed that the rate of disinfection of instances, novel formulations have been produced which are spores exposed to formaldehyde vapor increased as the VOL. 3, 1990 SPORICIDAL AGENTS 103

TABLE 5. Comparative sporicidal activities of some aldehydesa drolyze in water to produce an amino (=NH) group; their Sporicidal activity sporicidal activity is slower than that of the . Aldehyde Chemical formula Like the hypochlorites, activity of these compounds is Formaldehyde H * CHO Fair greater at acid than at alkaline pH. Cousins and Allan (42) Glyoxal CHO * CHO Only at 10% (not 2%) have demonstrated that sodium hypochlorite was the most Malonaldehyde CHO CH2 CHO Slight effective of five halogens against B. cereus and that B. Succinaldehydec CHO (CH2)2 CHO Slight subtilis spores were more resistant to all sporicides tested. Glutaraldehyde CHO * (CH2)3 * CHO High Generally, spores of Clostridium spp. are more susceptible Adipaldehyde CHO (CH2)4 CHO Slight to chlorine than are Bacillus spores (56). a Comments on sporicidal activity of aldehydes based in part on Boucher Freshly prepared hypochlorite solutions, buffered to about (28-30). pH 7.6, have a very rapid sporicidal activity (11, 116). h Some aldehydes have been reexamined against B. subtilis NCTC 8236 spores (Power and Russell, in press). Mixtures of 1.5 to 4% sodium hydroxide with sodium hy- c Gigasept contains both succinaldehyde and formaldehyde and is more pochlorite (200 ppm [200 pg/g] available chlorine) are much active (as a 10%o solution) than formaldehyde alone. more rapidly sporicidal than either sodium hydroxide or hypochlorite used singly (42). This could result from an effect of the alkali on the spore coat, thereby increasing temperature increased, that organic matter such as blood, hypochlorite penetration. Potentiation of sporicidal activity sputum, or soil reduced the rate of spore inactivation, and of hypochlorites is attained in the presence of methanol and that an increase in rate of kill occurred as the relative other alcohols (40, 51, 116), and buffering to pH 7.6 to 8.1 of humidity was raised to 50% with little increase above this alcohol-hypochlorite solutions produces powerful sporicidal value. There is no universal agreement about the effect of activity with optimum stability (51). Such solutions are still, relative humidity on activity. Russell (171, 172) reviewed the however, inactivated by organic matter. activity of formaldehyde and described work claiming that Iodine and iodophors. Iodine and iodophors (iodophores; no bactericidal effect occurs unless the humidity is 70% or literally, iodine carriers) are considered to be effective above. Confusingly, Hoffman (99) has reported that the bactericidal and sporicidal agents (207, 218). Iodine itself is aldehyde is quite effective even at humidity values of <50% sparingly soluble in cold, but more soluble in hot, water. and that formaldehyde generated from paraformaldehyde is Stronger solutions can be made in potassium iodide or in more active than an equivalent amount generated from alcohol. Iodine is less reactive chemically than chlorine and Formalin solution. Low concentrations of formaldehyde are is less affected by the presence of organic matter; neverthe- sporostatic. less, these effects depend on iodine concentration. The Other aldehydes. The sporicidal activity of aldehydes activity of low, but not of high, concentrations of iodine is other than glutaraldehyde and formaldehyde is equivocal significantly reduced. The sporicidal of iodine is also (Table 5). Borick et al. (26) stated that glyoxal was spori- pH dependent; at neutral and acid pH, diatomic iodine (12) is cidal, and Boucher (28, 30) has considered the efficacy of highly active and hypoiodous acid (HOI) also makes some various aldehydes. We have recently reexamined the effect contribution. At alkaline pH, activity is reduced, resulting of some aldehydes on B. subtilis NCTC 8236 spores (Power from the formation of the hypoiodide (OF) ion, which has and Russell, in press). Over a 5-h period at 22°C, 10% only a slight activity, and the inactive iodate (103), iodide butyraldehyde had no sporicidal action against the spores (I-), and triiodide (13) ions. A major problem with iodine is (109 CFU/ml at zero time), 10% glyoxal effected a 3-log that it is toxic and also stains fabric and tissues. reduction, and 8% formaldehyde produced a 4-log reduction. The iodophors consist of a loose complex of elemental Gigasept (Sterling-Winthrop, Surrey, United Kingdom) iodine solubilized by means of appropriate carriers which (containing succinaldehyde [butan-1,4-dial], formaldehyde, increase solubility while at the same time providing a sus- and 2,5-dimethoxytetrahydrofuran, pH 6.5) achieved, at 5%, tained-release reservoir of iodine (85) and stabilized with a 2-log reduction and, at 10%, a 5-log reduction. Malonalde- phosphoric acid. Suitable carriers consist of neutral poly- hyde was inactive, and the greatest rates of kill were mers such as nonionic surfactants and povidone (polyvi- obtained with 2% alkaline glutaraldehyde, activated Cidex nylpyrrolidone) and polyethylene glycols, which exhibit (Surgikos, Arlington, Tex.) and undiluted Sporicidin (glutar- surface-active properties and which, therefore, improve aldehyde, 2%; phenol, 7%; sodium, phenate, 1.2%; pH 7.4), wetting properties, thereby aiding in penetration into organic all of which produced reductions of at least 8 logs. Sporicidin soil. The iodophors have been reviewed by several authors was marginally the most active. (85, 171-173, 207, 218). The concentration of free iodine in Chlorine-releasing agents. Essentially, chlorine com- an iodophor is responsible for its bactericidal activity. In pounds can be considered as being of three types: chlorine many iodophor preparations, the carrier is a nonionic sur- gas, which is too hazardous for normal use; sodium and factant in which the iodine is present as micellar aggregates. calcium hypochlorites; and chlorine-releasing agents (55, When the iodophor is diluted with water, the micelles 218). Of these, the agents of choice are sodium hypochlorite, disperse and most of the iodine is slowly liberated. Dilution which contains up to 15% (wt/vol) available chlorine, and below the critical micelle concentration of the surfactant sodium dichloroisocyanusate, which slowly releases hy- results in iodine being in simple aqueous solution. Favero pochlorous acid (HOCl). The active species is undissociated (60) has made the important point that iodophors formulated hypochlorous acid, the hypochlorite ion (OCl-) being con- as contain much less free iodine than those siderably less so, and disinfection by chlorination is optimal formulated as disinfectants, which should contain 30 to 50 at around pH 6, at which dissociation of HOCI is minimal. mg of free iodine, or 70 to 150 mg of available iodine per liter. Chlorine compounds are bactericidal and sporicidal, al- Iodophors at high concentrations may be sporicidal over a though spores are more resistant than vegetative cells (22, wide pH range, but are much less potent than glutaralde- 23, 42, 55, 56, 147, 218). Activity of the hypochlorites is hyde; they do not stain and are nontoxic. greatly reduced in the presence of organic matter. Organic Peroxygens. The two important peroxygens are hydrogen N-chloro compounds, containing the =N-Cl- group, hy- peroxide (H202) and peracetic acid (CH3COOOH). 104 RUSSELL CLIN. MICROBIOL. REV.

The bactericidal and sporicidal properties of hydrogen ture effect for EtO concentrations of <880 mg/liter at tem- peroxide have long been known (4-6, 16, 17, 121, 215, peratures of <350C. However, a critical temperature is 224-226, 229-231) and are influenced by a variety of factors. reached for a particular concentration, after which an in- Of these, one of the most important is concentration, a low crease in concentration has no additional effect on the rate of concentration (6%, wt/vol) being bactericidal, but only kill of bacterial spores. At higher concentrations and tem- slowly sporicidal. However, at 25TC and levels of between 10 peratures about 32TC, the kinetics become zero order with and 20% (wt/vol), the concentration exponent is about 1.5 respect to concentration, with a Q10 value of 1.9. (173). Survivor curves of spores exposed to low peroxide (iii) Activity is water vapor dependent. Of all factors concentrations frequently exhibit a distinct shoulder. A influencing EtO activity, moisture vapor is the most critical "tailing" has also been observed (36), which has been variable (34, 58). The area is a complex one; the fact that attributed to the formation of spore clumps during treatment conflicting results were obtained by Phillips and Kaye (113- and the associated spore catalase, thereby destroying hydro- 115, 151, 152) and Gilbert et al. (75), on the one hand, and gen peroxide in the immediate vicinity. Temperature exerts Kereluk et al. (117) and Ernst (58, 59), on the other, is a a marked effect on sporicidal activity; at ambient tempera- reflection of the diverse test procedures used. The first group tures, peroxide is only slowly sporicidal, but the temperature recommended the use of a relative humidity (RH) of between coefficient (Q10) for each 10'C rise is about 2.5 (173). A factor 30 and 40%, whereas the latter presented data to show that that can influence activity of peroxide is its stability; it tends sporicidal efficacy increased with increasing RH. The low- to be unstable and its decomposition is increased by metals, level RH recommendations were based on work in which metallic salts, light, heat, and agitation, but it is compara- spores and their carrier materials were allowed to equilibrate tively stable in the presence of a slight excess of acid. with the RH of the test environment. The second group of Decomposition can be reduced by appropriate storage. workers were more interested in practical industrial applica- Peracetic acid is a bactericidal and sporicidal agent (13, 14, tions, and the spore test pieces were below equilibrium for 224). It decomposes ultimately to hydrogen peroxide, acetic the moisture content of the load against the RH of the acid, and , which at recommended in-use concentra- sterilizing environment. tions is toxologically safe. It is considered (13, 14) to be a In a model theory (59) put forward to explain these more potent sporicide than hydrogen peroxide, and its conflicting data, spores are characterized with respect to activity is reduced only slightly in the presence of organic their immediate environment and relative moisture content matter and is unaffected by the presence of catalase. Perace- as compared with the gross environment surrounding them. tic acid is more active at pH 5 than at neutral pH. The basis of this theory is that water molecules carry EtO to Ethylene oxide. Ethylene oxide [EtO; (CH2)20] exists reactive sites; thus, in an environment with a relatively low usually as a gas that is soluble in water, oils, rubber, and moisture content with respect to the reactive site, the most organic solvents. A major problem with its use is that dynamic exchange must be directed outward, i.e., from the it is inflammable when in contact with air, but in practice this spore. The movement of EtO gas is thereby impeded, and can be overcome by using mixtures of EtO with carbon the macromolecules of the cell are less amenable to alkyla- dioxide or fluorocarbon compounds. EtO is freely diffusible tion. When the moisture content of the immediate environ- and penetrates paper, cellophane, cardboard, fabrics, and ment increases, the equilibrium condition arises, which is some plastics but less readily through polyethylene. It is intermediate in effectiveness. As the environmental water unable to penetrate crystalline materials. content rises further, the dynamic movement of water is Early work (113-115, 151, 152) considered the chemical, directed towards the active site (the spore), the most ideal physical, and microbiocidal properties of EtO. Later studies situation in practice. In the case of a relatively dry spore and have fully supported these findings (34, 35, 39, 58, 59, 75, 99, low-RH environment, there is little exchange of moisture 117, 129, 159, 162, 171-173, 180) and have shown that the into and out of the spore, a situation very limiting for activity of EtO depends on several factors. sterilization in practice. As the water content of the spore (i) Activity is concentration and time dependent. As would and of the environment increases, a relatively wet spore and be expected, the higher the concentration of EtO, the more high RH are obtained, thus resulting in a zone of high rapid its sporicidal activity. For example, Phillips (151) moisture which would have a diluting effect on EtO gas, calculated values of 1/k (equivalent to the time in hours at reducing its availability to the spore (58, 59). This would be 250C required [t90%I to kill 90% of B. subtilis var. globigii the situation in those experiments (75, 113-115, 151, 152) in spores dried on cloth): values of 1/k were 7.2, 3.3, 1.6, 0.5, which the RH is above 40%, with an intermediate RH thus and 0.35 h for EtO concentrations of 22, 44, 88, 442, and 884 representing the optimal RH of ca. 32%, designated by these mg/liter, respectively. As the concentration of EtO in- workers. creases, obviously 1/k or t90% decreases. (iv) Activity depends on the type of organism. Contrary to These values demonstrate clearly that EtO is only slowly the situation with most liquid biocides, to which spores are bactericidal, even a high concentration (884 mg/liter), taking often several thousandfold more resistant than nonsporulat- 0.35 h to reduce the number of viable spores by 90%, i.e., 1 ing bacteria, spores are generally only some 2- to 10-fold log cycle. This slow rate of kill is an obvious disadvantage of more resistant to EtO gas than are vegetative organisms (35, the gas and is a property that is taken into account in 151, 152, 173). Spores of the thermophile B. stearothermo- providing suitable conditions for sterilization. philus and of certain other organisms may, in fact, be less (ii) Activity is temperature dependent. Sporicidal activity resistant to EtO than some vegetative bacteria such as S. of EtO is increased as the temperature is raised. Phillips aureus, Enterococcus faecalis, and Deinococcus radiodu- (151) calculated that the temperature coefficient (010 or Q10) rans. was 2.74 for each 10°C rise in temperature. The relationship IP-Propiolactone. ,B-Propiolactone is not widely used as a among concentration, time, and temperature is, however, sporicidal agent (39). It exists as a colorless liquid at room more complex than might be implied from this simple temperature, boils at 1630C, and may be vaporized in a statement. Ernst (58, 59) has shown that the death rate is special atomizer. P-Propiolactone is noninflammable, has logarithmic and that the Q10 of 2.74 describes the tempera- low penetrating powers, and is claimed to be carcinogenic. VOL. 3, 1990 SPORICIDAL AGENTS 105

TABLE 6. Neutralization of sporicidal and sporostatic activity Sporicidal agent Neutralizing agent Comment Glutaraldehyde Glycine ) Better then dilution or sodium bisulfite (could be toxic to germinating and outgrowing cells) Formaldehyde Glycine Hypochlorites Sodium thiosulfate Thiosulfate toxic to some streptococci; unlikely to be toxic to germinating and outgrowing spores Iodine and iodophors Sodium thiosulfate Hydrogen peroxide Catalase Very rapid effect of neutralizing agent Peracetic acid Dilution Ethylene oxide Dilution in recovery medium Specific neutralizer: guanine? Phenolsb and cresolsb Dilution or polysorbate Have high concn exponent; thus activity lost on dilution Organomercury compounds Sodium thioglycolate or cysteine' Thiglycolate might be toxic Chlorhexidine diacetate,b QACsb Lecithin + polysorbate Dilution inappropriate a Based on Russell et al. (176). b Sporostatic, but sporicidal at elevated temperatures.

Its bactericidal and sporicidal activity is a direct function Appl. Bacteriol. Tech. Ser., in press). It must be noted that of the concentration and the time and temperature at which glutaraldehyde interacts strongly with nutrient media (80, it is used (34, 35, 39, 99, 171-173). The temperature coeffi- 182) and that this may present an erroneous impression as to cient, Q10, in the range of -10 to +25TC is 2 to 3 (99). As with its apparently low sporostatic (or bacteriostatic) activity. EtO, however, the single most important factor determining Sporicidal evaluations may be of several types (8, 11, 36, its sporicidal is water vapor, and for optimum 71, 77, 85, 161, 173, 190, 202, 203, 227; Russell et al., in activity the RH should be kept above 70 to 75%. Again, press). Sporicidal activity can be tested against spores in however, it is not necessarily the atmospheric RH that is liquid medium or suspended on appropriate carriers. What- important, but the moisture content and location of water in ever method is adopted, and irrespective of whether quan- the bacterial cell. B. subtilis var. globigii spores equilibrated titative (survival counts) or qualitative (extinction) assess- to 98% RH are readily killed by P-propiolactone at an RH of ments are made, an appropriate method must be used to 45%, an RH at which the spores are not usually susceptible. determine spore survival (173). It cannot be emphasized too However, only a 2-log reduction (ca. 99% kill) is achieved strongly that adequate neutralization (quenching) of the test when spores equilibrated to 75% RH are exposed to this chemical must be achieved to prevent sporostasis occurring agent at 45% RH, and a small percentage of spores precon- in subculture media and, consequently, false-negatives (37, ditioned at 1% RH is thereafter very resistant to ,-propio- 78, 161, 173, 176). In brief, neutralization involves (i) diluting lactone at 75% RH (99). the biocide in the recovery medium to a level at which it Other gases. Other gases with sporicidal activity include ceases to have inhibitory activity, (ii) incorporating into the propylene oxide (reviewed in reference 173) and . The a that former is bactericidal and sporicidal, but less so than EtO, recovery medium neutralizing agent (antidote) specif- and is allowed to be used in the food industry. As with EtO, ically inactivates the biocide and is itself nontoxic to germi- its activity depends on concentration, on time and tempera- nation and outgrowth (177), or (iii) removing the biocide by t'ure, and especially on RH. means of a membrane filtration technique, followed by Ozone has bactericidal and sporicidal properties, but its washing the membrane in situ (with, if necessary, an appro- instability and other undesirable properties were considered priate neutralizing agent, e.g., with QACs [38, 181]), and (99) to render it unsuitable for use as a gaseous disinfectant. then placing it on the surface of a solid nutrient medium. However, more recent studies (63, 69) have demonstrated Suitable neutralization procedures for specific sporicidal the sporicidal activity of the gas, especially under acidic pH agents are summarized in Table 6. It must be added that conditions against spores (B. cereus, C. perfringens, and C. sodium thioglycolate, widely used as an ingredient of anaer- botulinum) of importance in food processing. obic culture media, may in fact inhibit vegetative cell devel- opment from treated bacterial spores (50, 97, 136). When placed in nutrient media held at the desired temper- RECOVERY AND REVIVAL OF INJURED SPORES ature, normal (control, untreated) spores usually germinate When exposed to chemical or physical agents, microor- very rapidly, a process complete within 30 to 60 min. Spores ganisms may be inhibited, sublethally injured, or irreversibly that have been damaged, however, invariably require long damaged, i.e., killed (7). In the laboratory, several types of periods to repair this injury (7, 49, 50, 90, 159, 173, 200, 201, tests are available for examining sporostatic and sporicidal 228), and for this reason it is prudent to prolong the incuba- activity. In essence, the sporostatic tests involve determin- tion period well beyond the usual period of, say, 48 h at ing the MIC of a chemical agent, i.e., the lowest concentra- (usually) 37°C; suboptimal incubation temperatures should tion preventing germination or outgrowth or both. Practical also be examined, as should the effect of composition and details can be obtained by consulting a forthcoming paper pH of the recovery medium (134). (A. D. Russell, B. N. Dancer, and E. G. M. Power, Soc. The revival of chemically injured spores may also be 106 RUSSELL CLIN. MICROBIOL. REV.

TABLE 7. Revival of bacterial spores exposed B. subtilis NCTC 8236 spores have been treated with glutar- to sporicidal agents aldehyde (which was then neutralized with glycine), washed .Posttreatment revival Refer- with buffer, and exposed to 20 mM NaOH for 10 min; the Sporicide.poricie procedures ence(s) release of protein was then determined chemically: only ca. 1 pFg was released by alkali treatment (49). It is, of course, Formaldehyde Heat activation (60-90'C), plating 201 possible that a small number of spores may possess a higher than average resistance to glutaraldehyde and become su- Glutaraldehyde UDS ± sonication, incubation in 76 GML, plating in TSA perdormant (86, 90) rather than damaged, so that they are able to germinate only under extreme conditions. Lysozyme Heat (50-90'C), dilution in GM or 76 may facilitate germination of damaged spores (90), although GML, plating in TSA this phenomenon applies mainly to thermally injured spores (see later results with hypochlorites, however). Gorman et NaOH or KOH, plating 49, 154 al. (76) reported that increased survivor counts of iodophor- treated B. subtilis spores were obtained following exposure Povidone-iodine Incubation in GML, plating in TSA 76 to lysozyme. a UDS, Urea plus DTT plus sodium lauryl sulfate; GM, germination Information on the revival of chemically damaged spores medium; GML, germination medium plus lysozyme; TSA, tryptose soy agar. is still, on the whole, lacking. From an academic or theoret- ical point of view, considerable data may be generated about mechanisms of sporicidal action (64, 65), and thus studies on achieved by specific procedures. It has been claimed that revival are always worthwhile. Likewise, any assessment of subjecting formaldehyde-treated spores of B. subtilis to a sterility must take into account conditions for repair of posttreatment heat shock at temperatures of 60 to 90'C injured, but still viable and potentially harmful, bacterial enables most of the supposedly killed spores to revive (200, spores. In the practical context, the studies from this labo- 201). A very small proportion of glutaraldehyde-exposed ratory with glutaraldehyde-damaged spores described above spores of various Bacillus spp. can be revived when, follow- have used severe revival conditions that are unlikely to be ing neutralization of glutaraldehyde with glycine, the spores encountered in practice; furthermore, an injured spore under are treated with alkali (49, 153). The rate of NaOH-induced such circumstances is effectively dead if it cannot germinate revival is ca. 10-6 (i.e., CFU per milliliter of glutaraldehyde- or outgrow. Nevertheless, there might be some small risk in exposed, NaOH-treated spores/unexposed, NaOH-treated overestimating the sporicidal efficacy of glutaraldehyde. spores), obviously a low value but one of potential impor- Spores have been recovered after 24-h exposure to the tance (49). Experiments designed to distinguish between dialdehyde, whereas the Association of Official Analytical germination and outgrowth in the revival process have Chemists test (8) recommends a 10-h exposure and much established that sodium hydroxide (range, 10 to 50 mM; shorter exposure periods are commonly encountered, par- optimum, 20 mM) added to glutaraldehyde-treated spores ticularly in the hospital environment where time is often at a increased the potential for germination. In contrast, B. premium. An additional point is that in our studies (76, 153) subtilis spores which are allowed to germinate before expo- only freshly activated glutaraldehyde solutions were used, sure to low concentrations of glutaraldehyde and then to whereas in practice older solutions are frequently used, with sodium hydroxide are inhibited at the outgrowth phase to a some deterioration occurring (155). Nevertheless, matters much greater extent than germinated spores treated with the should be kept in perspective, and Babb et al. (11) have dialdehyde without subsequent alkali exposure (153). So- stated that a 3-h treatment with 2% alkaline glutaraldehyde dium hydroxide can be replaced with potassium hydroxide should be sufficient for practical purposes to achieve a or, to a lesser extent, sodium bicarbonate. The use of 2% sporicidal effect, especially as bacterial spores are only (wt/vol) glycine (37) (Table 7) as an inactivator of glutaral- infrequently found on clean medical equipment. dehyde is of paramount importance in these revival studies. The situation with formaldehyde is potentially more Alkali-induced revival of spores exposed to another dialde- alarming. Although alkali treatment does not revive formal- hyde, glyoxal, has also been found with slight revival after dehyde-treated spores (Power et al., in press), the studies of exposure to Gigasept (containing succinaldehyde plus form- Spicher and Peters (200, 201) suggest that the sporicidal aldehyde) but not to formaldehyde alone (E. G. M. Power, activity of this monoaldehyde might well have been overes- B. N. Dancer, and A. D. Russell, Lett. Apple. Microbiol., in timated. Confirmation or rebuttal of their findings is awaited press). This interesting phenomenon could be related to the with interest. far more damaging effects on the spores of glutaraldehyde Injured bacterial spores might be of concern in food than formaldehyde, with a consequent greater potential for microbiology. Hypochlorites are used as sanitizers and alter revival of the former than the latter. germination responses of C. botulinum spores (65). In addi- Some revival of glutaraldehyde-treated spores can be tion, C. bifermentans spores are sensitized to lysozyme- achieved by means of a posttreatment heating (76, 154), but induced germination following treatment with hypochlorite the extent of this revival (maximum, two- to threefold (237). Exposure of spores to EtO or H202 may alter require- increase in viable count achieved at 57°C) is less than by ments for growth (64). Cook and Pierson (41) have pointed alkali treatment and also markedly less than that reported out that conditions used to enumerate spores in foods might with formaldehyde (201). Coat-removing agents fail to not be optimum for germination and outgrowth of all spores achieve any revival (154) despite reports that glutaralde- and that injured spores must be considered in this context. hyde-treated spores damaged by one such treatment are capable of germination (76). Lysozyme, either used before plating or incorporated into recovery media, is likewise SPOROGENESIS, SUSCEPTIBILITY, AND RESISTANCE ineffective, and a combination of NaOH and lysozyme has a Sporulation, germination, and outgrowth are complex slight, but noticeable, deleterious effect on colony counts processes in the overall life cycle of Bacillus and Clostridium (154). To determine whether alkali induces protein release, spp. Differing responses to biocides are shown at different VOL. 3, 1990 SPORICIDAL AGENTS 107

stages, and these aspects will be considered, as some useful TABLE 8. Onset of resistance to antibacterial agents information can be obtained about the mechanisms of spore during sporulationa resistance. Sporulation Sporulation at Agent stageresistanceat which stageresistancewhichis Comment Sporulation appears fully developed Sporulation is a multiphase process leading to the devel- Toluene Late stage III Early stage IV Early event opment of a spore from a vegetative cell. The stages in- Chlorhexidine Stage IV Stage V Intermediate volved (57, 118, 119) can be summarized as follows. Stage 0 Heat Stage V Stage VI Intermediate is the vegetative cell, stage I the presporulation phase (in Lysozyme Middle of Stage VI Late which DNA is present as an axial filament), and stage II is stage V the septation phase in which asymmetric cell formation Glutaraldehyde Late stage V Stage VI corm- Very late event occurs. Engulfment (encystment) of the forespore takes pleted place in stage III and cortex formation between the inner and a Based on Power and Russell (in press) and Shaker et al. (188). outer forespore membranes commences in stage IV, with synthesis of spore coats, dipicolinic acid, and uptake of Ca2+ in stage V. Spore maturation occurs in stage VI, with the and Russell, in press). Nevertheless, in general terms, the coat material becoming more dense and refractility increas- idea of attempting to correlate resistance with a specific ing. Lysis of the mother cell and liberation of the mature components) of the spore coat is an attractive one and spore take place in stage VII. Clearly, there are several should be subjected to further experimentation. stages at which antibacterial agents could act or, conversely, The increased resistance occurring during sporulation may when resistance to such agents could arise. Sporulation thus be related to the stage of spore development (Table 8). (Spo-) mutants which are unable to develop beyond a In many instances, the chemicals studied, e.g., xylene, genetically determined point (98, 108, 109) are of consider- toluene, or benzene, have been organic solvents rather than able value in correlating structural changes, biochemical preservatives or disinfectants. Resistance to chloroform and characteristics, and susceptibility or resistance to specific to phenol, however, develops late in the sporulation process biocides. A practical consideration of these aspects is being (12, 130), and that to methanol and ethanol occurs at the published elsewhere (Russell et al., in press). same time as resistance to other alcohols, such as octanol Disinfectant-induced structural changes in fully developed and butanol. Alcohol-resistant sporulation mutants of B. spores have been described (123, 168, 189), but these have subtilis can sporulate in the presence of alcohols at a not been fully related to their biochemical effects on sporu- frequency of 30 to 40% (24). lating cells. Thus, the mechanism of action (see later section) In conditional spore cortexless mutants of B. sphaericus of many sporicidal agents is still often poorly described. In deficient in the synthesis of meso-diaminopimelic acid (Dap), contrast, the mechanisms of spore resistance to biocides are the muramic lactam (and hence cortex) content increases better understood, and these aspects will be considered with an increase in exogenous meso-diaminopimelic acid here. (103, 104). Characteristic spore properties have been found Resistance of bacterial spores (209) can be examined by (i) to be associated with different amounts of cortex; e.g., ca. comparing the response of wild-type and Spo- mutants; (ii) 25% of maximum cortex content is necessary for the spore to using other mutants, e.g., conditional cortexless mutants of present resistance to octanol but ca. 90% is necessary to B. sphaericus (103, 104); or (iii) comparing "normal" and show heat resistance. Such Dap- mutants might thus be coatless forms of a spore. useful in studying mechanisms of spore resistance to bio- Development of resistance to biocides and antibiotics cides, although as pointed out by Waites (224), changes during sporulation (82, 83, 130, 210) has been known for other than variations in cortex development might occur some time. Experiments designed specifically to associate elsewhere in the spore which must be considered before changes in cell structure with altered responses to biocides ascribing resistance solely to the cortex. can yield useful information in this area. There is a need, Probably the most detailed approach to studying resis- however, to correlate these structural changes more accu- tance of spores has involved the use of spore coatless forms rately with biochemical changes in the spore. Useful mark- (46, 62, 63, 69, 82, 88-91, 93, 124, 126, 132, 189, 194, 212, ers for monitoring the development of resistance are toluene 224-228, 231, 237). Methods of removing one or both spore (resistance to which is an early event), heat (intermediate coats have been described in detail by Nishihara et al. event), and lysozyme (late event) (108-111). In studies with (141-143). Coats may be extracted by using 2-mercaptoeth- a wild-type B. subtilis, strain 168, and its Spo- mutants, we anol, sodium lauryl (dodecyl) sulfate, dithiothreitol (DTT), have demonstrated (156a, 188) that resistance to chlor- and urea. Treatments consist of urea plus DTT plus sodium hexidine occurs later than that to toluene and at about the lauryl sulfate, urea plus DTT, urea plus 2-mercaptoethanol, same time as heat resistance, whereas glutaraldehyde resis- and sodium lauryl sulfate plus DTT. Of these treatments, tance is a very late event, occurring after the development of urea plus DTT plus sodium lauryl sulfate is usually consid- lysozyme resistance (Table 8). Some 12 or so polypeptides ered the most satisfactory. Both lysozyme and nitrous acid are found in the spore coat of B. subtilis (108-111); these are or sodium nitrite are effective against coatless, but not synthesized at different times and are incorporated into the normal, spores, although pretreatment of the coatless spores spore at stages V and VI. It has been suggested (108) that with glutaraldehyde reduces the extent of this activity con- one polypeptide of molecular weight 36,000, which is formed siderably (82). The role of the spore coat in resistance of very late in sporulation, may have a direct role in conferring spores to various antibacterial agents is summarized in Table resistance upon the spores. The development of glutaralde- 9. Hydrogen peroxide itself will remove coat protein from C. hyde resistance, however, is unlikely to result from the bifermentans (231); however, removal of coat protein by deposition of specific spore coat proteins because of the DTT before spore exposure to peroxide markedly increases highly reactive nature of the dialdehyde molecule (Power its lethal effect, whereas B. cereus spores are much less 108 RUSSELL CLIN. MICROBIOL. REV.

TABLE 9. Mechanisms or site of resistance of bacterial Germination spores to chemical agents Activation is a treatment resulting in a spore which is Antibacterial Spore Comment agent component poised for germination but which still retains most spore properties; activation is thus responsible for the breaking of Alkali Cortex dormancy in spores, but is reversible. In contrast, germina- tion itself is an irreversible process and is defined as a change Lysozyme Coat(s) 1 to a Hypochlorites Coat(s) UDS spores highly sensitive of an activated spore from a dormant metabolically Glutaraldehyde Coat(s) active state within a short period of time. Iodine Coat(s) The first biochemical step in germination is the biological trigger reaction. This initiation process can be induced by Hydrogen peroxide Coat(s) Varies with strain metabolic or nonmetabolic means, although it is now gener- ally believed that the trigger reaction is allosteric in nature Chlorhexidine Coat(s) UDS spores more sensitive rather than metabolic, because the inducer does not need to than "normal" spores be metabolized to induce germination. Initiation of germina- tion is followed rapidly by various degradative changes in Ethylene oxide Coat(s) Exact relationship unclear the cell, leading within a short period of time to outgrowth. Octanol Cortex | Dap- mutants of B. sphaeri- These changes include (87, 217) (i) a decrease in heat Xylene Cortex J cus more sensitive resistance accompanied by changes in staining properties, (ii) a decrease in refractility whereby phase-bright spores a Dap, meso-Diaminopimelic acid. (Fig. 2a and b) become phase dark (Fig. 2b and c), (iii) a decrease in dry weight, and (iv) a decrease in optical density, a comparatively late event in germination (205), although it is a widely used method for measuring germination. Inhibition affected (226). The spore coat is thus likely to confer a and control of spore germination are important consider- protective effect against peroxide to the former but not to the ations in many fields, including food preservation (74, 193), latter spores, thereby demonstrating that varying responses although dormancy may be a problem (86). occur with different sporeformers and that the response may Several antibacterial agents are known to inhibit germina- be associated with different composition and structure of the tion. These include alcohols, aldehydes, phenols and spore coat(s). Spores treated with urea plus DTT plus cresols, parabens, sorbic acid, and mercuric chloride (2, 3, sodium lauryl sulfate are highly susceptible to glutaralde- 47, 66, 68, 100, 128, 148, 149, 156, 166, 168, 171, 172, 181, hyde, iodine, hydrogen peroxide, ozone, and chlorine (63, 190, 193, 195, 198, 219, 220, 223, 234; B. M. Lund, Ph.D. 76, 82, 174). Thus, even with agents that are known to be dissertation, University of London, London, England, actively sporicidal, the coats play a role in limiting intracel- 1962). This inhibition (Table 10) occurs at concentrations lular penetration. that are closely related to those that inhibit the growth of Nevertheless, the role of the spore coats in resistance to vegetative bacteria. EtO is unclear. In B. subtilis, removal of the coats increases The effects of inhibitors of spore germination may be spore sensitivity (129). However, resistance to EtO of spores reversible. This is apparent from the results of studies with of B. cereus strain T pretreated with alkaline DTT remains phenols (128, 148, 149, 181), formaldehyde (219), alcohols unchanged (46). Furthermore, B. subtilis 4673 (a mutant of (220), and parabens (157, 234). These findings suggest a fairly strain 4670) with defective coats and outer coat layers loose binding of these agents to a site(s) on the spore surface thinner and more diffuse than 4670 is more resistant to EtO since mere washing is often sufficient to dislodge the inhib- than is 4670. Conversely, strain EV15 which overproduces itor. coat material, thereby possessing an abnormally thick mul- Mercuric chloride is a powerful inhibitor of the germina- tilayered coat, has an exceptionally high resistance to EtO tion of spores of C. botulinum type A (2) and ofBacillus spp. (46). On the other hand, these findings imply that the (92, 100, 223). It appears to inhibit some reactions in expected increased permeability to EtO in strain 4673 does germination before the loss of heat resistance but not the not occur and, on the other, that increased resistance to EtO subsequent release of peptidoglycan (223). In contrast, an is associated with excessive coat production. EtO is a organomercurial compound, phenylmercuric nitrate, has comparatively small molecule, but molecular size appears to been shown (148, 181) to have little effect on the germination be of little consequence where coat impermeability is con- of B. subtilis spores but a pronounced inhibitory effect on cerned. For example, H202 (molecular weight, 34), ozone outgrowth. (03; molecular weight, 48), and (C102; Germination (Ger) mutants of B. subtilis 168 deficient in molecular weight, 67.5) are all small molecules, yet spore the initiation of germination 135, 184), could be of value in coats are considered a primary protective barrier to their studying the mechanism of action of antibacterial agents but entry (63, 69, 70, 123). do not, as yet, appear to have been studied in this context. The spore coat appears to act as a permeability barrier to Glutaraldehyde exerts an effect early in the germination chlorine (123, 224, 226, 237), since coatless spores are process (153, 156). This belief is based on several experi- rendered more permeable to hypochlorites. Chlorine will mental approaches, a very recent one (156a) involving the itself remove coat protein and allows lysozyme to initiate effect of the dialdehyde on the uptake of L-[14C]alanine to B. germination (237). Sodium hydroxide increases the perme- subtilis spores. This germinant is considered to act by ability of bacterial spores to germinants, and the potentiation binding to a specific to the spore coat (184), and of hypochlorite action by sodium hydroxide (42) may be the once spores are triggered to germinate, they are committed result of the effect of the alkali on spore coats from which irreversibly to losing their dormant properties (205). The protein is removed (93), although the cortex is alkali resis- aldehyde could inhibit germination by (i) reducing L-alanine tant (123). uptake as a consequence of competition for binding sites on VOL. 3, 1990 SPORICIDAL AGENTS 109 (a) (b) (c)

FIG. 2. Changes during spore germination examined by phase-contrast microscopy. (a) Mature, phase-bright spores; (b) development of phase-dark forms; (c) germination complete with full conversion to phase-dark cells. the spore, (ii) preventing passive diffusion of L-alanine into vent the trigger reaction by some unexplained means. At the spore, (iii) sealing the spore surface, or (iv) inhibiting the higher glutaraldehyde concentrations (01. to 1%, wt/vol), L-alanine-induced trigger reaction of germination by a later, uptake of L-alanine is significantly reduced, presumably the as yet unexplained, mechanism (Fig. 3a to d, respectively; result of a sealing effect by the aldehyde on the spore Table 11). In our experiments, despite earlier claims to the surface. Spores do not concentrate L-alanine and uptake contrary (112, 238-242), D-glucose has no significant effect proceeds rapidly without the necessity for an energy-depen- on L-alanine uptake. These other workers, however, calcu- dent active transport system, demonstrating that the dor- lated the binding affinity of glucose solely on the loss of heat mant spore is freely permeable to the amino acid, which resistance and turbidity of germinated spores rather than by enters by simple diffusion (53, 154). direct methods. Glutaraldehyde-treated spores retain their The effects of inhibitors of germination have been widely refractility, having the same appearance under the phase- reported, but it is often not known at what stage of germi- contrast microscope as normal untreated spores (Fig. 2a), nation an inhibitor is active (193). Because of the nature of even after subsequent incubation in germination medium. the germination process, the only types of antibacterial The observation suggests that inhibition occurs very early agents that are effective are those that inhibit the trigger in the germination process. At concentrations up to 0.1% reaction and those that prevent the degradative processes. (wt/vol), both acid and alkaline glutaraldehyde inhibit ger- Many antibacterial agents are known to affect the optical mination, but not L-[14C]alanine uptake, and therefore pre- density changes that occur in germination. A decrease in

TABLE 10. Inhibitors of germination and outgrowth SPORE Process Inhibitor Comment Germination Glutaraldehyde Probably inhibits trigger mechanism Sorbic acid Inhibitor of trigger mechanism? Formaldehyde, alcohols Diverse group, phenols, parabens probably different mercuric chloride J sites of action Sodium thioglycolate Caution needed with recovery media L-AI Outgrowth QACs, chlorexhidine No or little effect on EtO, organomercurials germination Hypochlorites I Trigger for Glutaraldehyde Even more effective at these stages germ i nation Sorbic acid Multiple sites of inhibition FIG. 3. Possible sites of action of an inhibitor of the trigger mechanism in spore germination. 110 RUSSELL CLIN. MICROBIOL. REV.

TABLE 11. Effect of glutaraldehyde on the germination manner when germination is carried out in a medium that trigger mechanism supports vegetative cell growth. After germination, germi- nated spores become swollen and shed their coats to allow Process Effect of glutaraldehyde the young vegetative cells to emerge, elongate, and divide. Decrease in optical Low concn (<0.1%) inhibitory, con- Of the macromolecular biosynthetic processes occurring density siderably below sporicidal levels after germination, RNA synthesis is the first, followed (2%) closely in Bacillus spp. by the onset of protein synthesis, with DNA synthesis occurring some time later. During L-['4C]alanine binding to Inhibition, but only at high aldehyde outgrowth, all types of RNA are synthesized. Cell wall spores concn synthesis commences after RNA and protein but before Phase darkening of Low concn (<0.1%) prevents (see DNA and coincides with swelling of the germinated spore. spores Fig. 2) Several antibacterial agents act at the outgrowth rather than the germination stage (Table 10). These include QACs, Outgrowth of previously Even more inhibitory than vs germi- organomercurials, chlorhexidine, and EtO (35, 148, 181, 190; germinated spores nation Lund, Ph.D. dissertation), the first three of which are sporostatic agents, with EtO a sporicidal compound. QACs bind strongly to spores, and simple washing procedures will optical density is a late event in germination and is not not remove them (38). QAC-treated spores which are mem- suitable for studying the initial reactions (193). Thus, brane filtered are still prevented from undergoing outgrowth whereas many compounds are likely to prevent the degra- when transferred to an appropriate growth medium (38, 181), dative processes, it is unclear whether the trigger reaction is and a neutralizing medium must be used in conjunction with also affected. Even a procedure involving a short period of membrane filtration. exposure to the inducer, e.g., L-alanine, followed by moni- The parabens and similar substances inhibit germination at toring of the fall in optical density is considered to be sporostatic concentrations. Outgrowth is prevented at higher inappropriate, as is one involving the release of 45Ca (205). concentrations. High concentrations of hypochlorites are Suitable methods are the direct one, described above, in- necessary to prevent spore germination, whereas moderate volving the uptake of labeled L-alanine, and one in which concentrations markedly retard outgrowth and low concen- spores are exposed to L-alanine for a very short period of trations have only slight effects on either (237). Sublethal time. The reaction is then stopped by adding an excess of its concentrations of EtO inhibit outgrowth but not germination competitive inhibitor, D-alanine. The commitment to germi- (159), and resistance of spores to EtO does not decrease nation is then measured by counting the conversion of during germination (46, 47). Hydration of the spore core and phase-bright spores (Fig. 2a) to phase-dark spores (Fig. 2c). alteration of spore coat layers do not therefore appear to be This technique has been used to study the effect of sorbate, linked to an increased susceptibility. Even spores exposed to an effective inhibitor of germination (198). Busta and his high EtO concentrations can germinate freely under a vari- colleagues (21, 198) have concluded that sorbic acid does not ety of conditions but will not outgrow (47), but asparagine compete with L-alanine for a common binding site on the acts as a germinant for untreated but not EtO-treated spores. bacterial spore, so that inhibition occurs after germinant A former food preservative, sodium nitrite, has been the binding (41). subject of heated debate as to how it exerts its antimicrobial Alcohols inhibit the L-alanine-initiated germination of B. activity (173). It does not affect spore germination and in fact subtilis spores, suggesting that this inhibition results from an induces germination, but only at high concentrations (2, 3, interaction of a hydrophobic region in or near the L-alanine 125). Nitrite inhibits postheating germination or outgrowth receptor site on the spore with the hydrophobic group on the or both, heat-injured spores being rendered more susceptible alcohol (242). Such interaction is presumably of a weak to the salt (105). nature, because (as pointed out earlier) the inhibition of It is apparent from this section and the preceding one that germination by alcohols is reversible. Unfortunately, only an most sporostatic compounds inhibit either germination or optical density technique was used in these studies. The heat outgrowth. (An exception to this general statement is glu- activation of C. perfringens spores at a temperature range of taraldehyde, low concentrations of which inhibit both pro- 70 to 80'C in water is enhanced in the presence of alcohols cesses [154]). What is not clear is why this should be so. (43, 44). The concentration of a monohydric alcohol to There has been little basic research to explain why one produce optimum spore activation is inversely related to its compound should, for example, inhibit the degradative pro- hydrophobic character. cesses associated with germination, whereas another com- Other inhibitors of germination include sodium bicarbon- pound has no effect at this site but inhibits the later stage of ate (15, 45) and cyclic polypeptide antibiotics (96). Antibiot- outgrowth. Figure 4 summarizes the effects of antibacterial ics are outside the scope of this paper, but the experimental agents on germination and outgrowth, as well as detailing approach involving morphological changes and inhibition of stages during sporulation at which specific resistances de- macromolecular syntheses has yet to be applied to many velop. biocides. B. brevis Nagano wild type produces the gramicidin S, which inhibits germinating spores (138). A OVERCOMING SPORE RESISTANCE particularly interesting property of this organism is that Bacterial spores can pose a problem insofar as the activity germination-initiated spores retain their resistance proper- of chemical agents is concerned. It is obviously essential to ties (48), and it is likely that this property could be studied use appropriate concentrations at the optimum pH for a further with a range of biocides. sufficient period of time to ensure a sporicidal effect. There are, however, means available for achieving the same, or an Outgrowth enhanced, response. These involve a combination of a Outgrowth is defined as the development of a vegetative chemical and a physical process or of two chemical agents cell from a germinated spore and takes place in an orderly (1). VOL. 3, 1990 SPORICIDAL AGENTS 111

Glut, -SPORE----... IGlut?. TABLE 12. Mechanisms of action of some chemical agents Lys . t Trigger.*-- SA? Antibacterial Site or mechanism CHA- do Degradative.. Other germination agent of action Tol- processes inhibitors? Alkali Inner spore coat Chlorine compounds Cortex CELL CULTURE GER INATION Ethylene oxide Alkylation of core protein and DNA Glutaraldehyde Cortex Hydrogen peroxide Spore core? Several. , V AsGlutO0 Lysozyme Cortex (1, 1--4 links) inhibitors? Glt Nitrous acid Cortex (at muramic acid residues)

OUTGROWTH FIG. 4. Summary of possible effects of some antibacterial agents alcohol) kills spores in 4 h (208). Even combinations of local on germination and outgrowth and of the development of resistance anesthetics and preservatives are claimed to be sporicidal during sporulation. Glut, Glutaraldehyde; SA, sorbic acid; Tol, (1). toluene; CHA, chlorhexidine diacetate; Lys, lysozyme; EtO, eth- Clearly, no comprehensive studies have been undertaken ylene oxide. to determine what factors are involved in designing a process with enhanced sporicidal activity. Also, reasons for any synergism are often totally inadequate. This aspect is one The bactericidal and sporicidal activity of a biocide in- that could, with benefit, be addressed. creases with increasing temperature. For example, the tem- perature coefficient (0) per 1C rise in temperature for the phenolic agent chlorocresol is 1.1 (33). If the temperature is MECHANISMS OF SPORICIDAL ACTION increased from 20 to 1000C, then assuming that 0 is the same A considerable amount of information is available about over the entire temperature range, the activity increases by the ways in which bactericidal agents affect nonsporing >2,000-fold (0100-20 = 1.180 = 2,104). Use of this principle bacteria (175). In contrast, mechanisms of sporicidal activity was, until 1988, made in the United Kingdom, where "heat- are poorly understood (236). The major reason for this is ing with a bactericide" was one official (pharmacopoeial) undoubtedly the complex nature of the bacterial spore, to method of sterilizing certain parenteral and ophthalmic prod- which may be added the possibility that an antibacterial ucts. Differences between acid and alkaline glutaraldehyde compound might have more than one actual or potential site forms disappear at temperatures above about 400C (28-30, of action. While contributing to the overall lethal effect, this 213). A process that has been promising results is the use of possibility can complicate still further the unraveling of the saturated steam at subatmospheric pressure in the presence mechanism of sporicidal action. of formaldehyde (173). The complicated effect of heat and The spore does present several sites at which interaction sodium nitrite has been described (105) and reviewed (173). with an antibacterial agent is possible, e.g., the inner and Acid heat treatment is an important means of controlling outer spore coats, cortex, spore membranes, and core (Table spores in food, spores being more susceptible to heat at low 12). Interaction with a particular site need not necessarily pH (20). Spores can, in fact, exhibit a base exchange imply, however, that this is associated with death of the behavior which will reduce, restore, or enhance their ther- spore or that there is only one site or target in the spore that mal resistance. Sensitization involves converting them to the must be inactivated. Practical considerations have recently hydrogen (H) form, which can be transformed into the been described (Russell et al., in press). resistant calcium (Ca) form by treatment with calcium ace- Data on uptake of an antibacterial compound to bacterial tate at pH 11. H-form spores are also more susceptible to cells are often considered a useful starting point in examining propylene oxide but less so than the Ca form is to glutaral- the of the compound. Studies on the uptake dehyde (210, 214). of glutaraldehyde to different types of bacteria (156) have Another physicochemical process is the use of glutaralde- shown that E. coli, B. subtilis vegetative cells, and S. aureus hyde with ultrasonics (28-31, 192). Although ultrasonic bind more aldehyde than do resting B. subtilis spores. waves themselves possess little sporicidal activity (and Uptake increases during spore germination and outgrowth have, in fact, been used to separate spores and vegetative but is less than to vegetative cells. The surface of bacterial cells), they have been claimed to potentiate the sporicidal spores is hydrophobic in nature (54). Low concentrations of activity of acid glutaraldehyde at 60'C and of hydrogen both acid and alkaline glutaraldehyde increase this surface peroxide but not of iodophors. The QAC benzalkonium hydrophobicity (156), presumably as a consequence of the chloride still had a poor sporicidal effect (31). A synergistic extensive interaction of the dialdehyde with outer layers of effect of hydrogen peroxide used in conjunction with UV cells and spores (19, 84, 140, 160, 211). The greater spori- radiation has been shown to occur (17, 230). cidal activity of the alkaline form is not reflected by the Combinations of chemical agents have also been studied. uptake patterns but it is likely that acid glutaraldehyde Examples have already been provided of hypochlorites and resides at the cell surfaces, whereas the alkalinating agent, methanol (40, 51) and of glutaraldehyde with nonionic (28- sodium bicarbonate, assists in the increased penetration of 31) or anionic (81) surfactants or with inorganic cation- the alkaline form into the spore (120, 137). The major initial anionic surfactant combinations (81). In many instances, effect of bicarbonate is believed to be on the outer layers of however, the underlying reasons for this potentiation remain spores of bacterial cell walls (79-81, 84, 137), although it will obscure. Glutaraldehyde has also been combined with phe- also inhibit germination of Bacillus spores (15, 45). Glutaral- nol plus phenate, the combination being claimed (106, 107) to dehyde is thus likely to seal the outer layers of spores, an have an enhanced sporicidal action. Alcohol is not spori- action that would also be of importance in inhibiting spore cidal, and only high concentrations (9%) ofhydrochloric acid germination (154), with penetration at alkaline pH into the have this property, whereas acid alcohol (1% HCl in 70% spore. Glutaraldehyde combines with amino acids (94, 182, 112 RUSSELL CLIN. MICROBIOL. REV.

235) and has been found to interact strongly with the cortex Spores of C. bifermentans produced on different media and spore protoplast, the latter prepared by Fitz-James' react differently to hydrogen peroxide, the more resistant technique (61). Interaction with the cortex might be respon- types having a thicker cortex and smaller protoplast (227, sible for the sporicidal action of the aldehyde. Penetration 229). Peroxide has a marked effect on spore structure (123, and reaction of glutaraldehyde with components of this layer 231), the cortex becoming depleted or absent and the ribo- may be assisted by the action of divalent cations (81, 84). somes becoming disordered. Other antibacterial agents also interact with the outer Taken as a whole, the above findings suggest that hydro- spore layers. Chlorhexidine diacetate increases spore hydro- gen peroxide has an effect on the spore coats in some phobicity (189) but is not sporicidal (188, 190) unless used at organisms but that this in itself is insufficient to explain its high concentrations at high temperatures (77). As with sporicidal activity. Its major effects are undoubtedly on the QACs, therefore (38), it is likely that this cationic agent cortex and core. The evidence to date implies tentatively combines strongly with spore coats, but is unable to pene- that the core is the major site of action, but further studies trate into the spore (169). are needed to substantiate this conclusion. Hypochlorites solubilize the cell walls of nonsporing bac- Of the other sporicides, the mechanism of sporicidal teria and the spore integuments of B. megaterium spores action of only one (EtO) has been examined in detail. The (167). Separation of spore coats from cortex, followed by mode of action of iodine has, surprisingly, been little studied sequential dissolution of spore layers, has been described (173). It is considered to bind to bacterial protein (207, 208), (123). Bacillus and Clostridium spores exposed to hypochlo- but this vague attribute does little to explain how it kills rites leak dipicolinic acid (56, 62, 123), suggesting an in- spores. Formaldehyde is considered (208) to be sporicidal crease in spore permeability that can also be achieved by because it can penetrate into the interior of the bacterial heat alone (18). The spore coat appears to act to some extent spore. This monoaldehyde is an extremely reactive chemical as a permeability barrier to chlorine (123, 224-226, 237), (173, 179), combining with protein, RNA, and DNA, but the since the removal of protein from spore coats renders spores reasons for its sporicidal action remain somewhat obscure. more susceptible to hypochlorites. These chlorine com- EtO is an alkylating agent believed to inactivate bacterial pounds will themselves remove coat protein, thereby allow- spores by combining with various groups in proteins and ing accessibility of cortex to lysozyme, which initiates nucleic acids (34, 35, 173, 174). B. subtilis spores exposed to germination (67, 237). Pretreatment of spores with sublethal EtO release considerably greater quantities of DNA, RNA, concentrations of chlorine renders the cells more susceptible protein, and dipicolinic acid than do untreated spores (129), to mild heating (56). This effect may result from an alteration but EtO is not mutagenic to bacteria or spores (75, 99), of spore cortex (237) since the cortex is believed to control unlike liquid sulfur mustard, an alkylating agent which is also spore response to high temperatures (89-91). The cortex mutagenic. may therefore be the major site of chlorine action, particu- Exposure of spores to trichloroacetic acid alters their larly since the removal of spore coats does not affect spore viability and germinability and their response to heat and viability. alkali but not to lysozyme (191). Trichloroacetic acid could The mechanism of sporicidal action of hydrogen peroxide help provide useful data about mechanisms of action of other has also been widely studied (4-6, 13, 14, 16, 17, 93, 121, agents. 166, 173, 174, 204, 215, 224-226, 229, 231). Hydrogen peroxide removes coat protein from C. bifermentans, but MEDICAL AND OTHER USES OF CHEMICAL coat protein removal (by DTT) from spores prior to peroxide SPORICIDES treatment markedly increases its effectiveness, although B. cereus is affected to a lesser extent. Sublethal levels of Previous sections have dealt with the spectrum of activity peroxide increase the germination rate in C. bifermentans. and mechanisms of action and of bacterial resistance. This Exposure of peroxide-treated spores to monovalent cations section will consider some uses of chemical sporicidal agents or to increasing pH results in a complete loss of their (see also references 73, 175, and 183). refractility (231). H202 increases the lysis of spores of C. bifermentans in the presence of certain divalent cations such Sporicidal Agents as Cu2+, but the effect with other spores is less marked (16, 17). Although C. bifermentans and B. subtilis var. niger Glutaraldehyde, one of the most potent sporicidal agents, spores take up Cu2+ at about the same rate, only the spore is extensively used in the leather tanning industry and in protoplasts of the former bind these cations. tissue fixation for electron microscopy and has numerous Activation of peroxide to hydroxyl radicals (-OH) is nec- biochemical applications (11, 84, 179). In the microbiological essary for sporicidal action, which would explain the syner- context, the dialdehyde has chiefly been used for the chem- gistic effect noted with the combined use of hydrogen ical sterilization of medical equipment which cannot be peroxide and UV radiation (17, 228, 230). DTT-treated sterilized by physical methods (84, 163, 185, 186, 216, 222). spores of C. perfringens are much more susceptible to The main advantages claimed for its use as a chemosterilizer peroxide-induced lysis in the presence of Cu2+ ions than are are (i) its broad spectrum of activity, especially good spori- untreated spores (4), and, significantly, this lysis is reduced cidal properties; (ii) its activity in the presence of organic by -OH scavengers. Peroxide and Cu2+, alone, do not matter; (iii) its rapid antimicrobial action, although spores produce lysis of cortical fragments but in combination in- are considerably less susceptible than nonsporing bacteria; duce lysis. It has been suggested (4) that peroxide may react (iv) its noncorrosive action towards metals, rubber, lenses, with Cu2+ bound to cortex peptidoglycan, thereby generat- and most materials, although some formulations may not ing -OH radicals. These would be formed at the region near fulfil these criteria (9, 11); (v) its lack of harmful effects on the germ cell wall and would be responsible for causing cement or lenses of bronchoscopes, cystoscopes, or tele- protoplast lysis. DTT-treated C. perfringens spores undergo scopes; and (vi) its ease of use. Nevertheless, its pungent germinationlike changes followed by lysis when exposed to and irritating odor to personnel over long periods of time is peroxide-generating systems (5). a distinct disadvantage (206). Rittenbury and Hench (164) VOL. 3, 1990 SPORICIDAL AGENTS 113 and Haselhuhn et al. (95) recommended glutaraldehyde for much less potent than thermal sterilization methods, espe- the cold sterilization of hemostats, cystoscopes, food con- cially autoclaving, but often find a use in sterilizing thermo- tainers, and anesthesia equipment. The aldehyde has also labile equipment; (ii) it is somewhat arbitrary to consider been found to be completely satisfactory for the routine only their sporicidal activity, since many of the biocides are sterilization of urological instruments and endoscopes (133, also used in other environments, e.g., as disinfectants, or as 145) and has proved highly effective for the rapid and safe decontaminants in specific areas as with materials contam- disinfection of gastrointestinal endoscopy equipment (216). inated with mycobacteria, human immunodeficiency virus, Despite inadequate sterilization with glutaraldehyde, be- or hepatitis B virus. cause of the short time periods often used in hospital practice, infection transmission appears to be rare, presum- Inhibitors of ably because very few potentially pathogenic spores are to Germination and Outgrowth be found on cleaned endoscopes (9, 11). Bovallius and Anas Antibacterial agents that are not sporicidal but instead (32) demonstrated the effectiveness of vapor-phase glutaral- inhibit germination or outgrowth or both have uses that are dehyde for surface disinfection against sporing and nonspor- different from sporicides. Sporeforming bacteria are of par- ing bacteria. In spite of its low volatility, it was more ticular concern in food products (74, 127), especially when effective than formaldehyde. they are capable of surviving food-processing treatments, of Formaldehyde is used as a solution and in vapor-phase causing food spoilage, and of being foodborne pathogens, form. In the liquid phase, formaldehyde is used as a disin- e.g., C. perfringens, C. botulinum, and B. cereus (41, 69). fectant and as a general farm disinfectant (180). Low- Because of changes in the food itself (palatability and temperature steam (without formaldehyde) for 10 min at nutritional aspects), it is often impossible to destroy all 730C is probably the most suitable method for disinfecting spores that might be present. Consequently, specific antimi- cystoscopes between patients (11). Longer periods of use of crobial agents are often included to inhibit growth from low-temperature steam with formaldehyde are required for spores. Sodium nitrite delays, but does not prevent, botuli- sterilizing laparoscopes and arthroscopes, but this damages nal outgrowth (41, 105), but its potential to humans most flexible fiber-optic endoscopes (11). is now well known (196, 197). Methyl and propyl parabens Chlorine-based products are used as food sanitizers, many are commonly used in the food industry as preservatives, of which are designed to control bacterial spores (70). with the propyl ester more effective in inhibiting C. botuli- Hypochlorites are used as disinfectants in the dairy industry, num growth and toxin production (171). Sorbic acid is a for the disinfection of farm buildings (180), and as disinfec- weak lipophilic acid widely used as a food preservative (127, tants in hospitals and food establishments (23). They have 168); it inhibits botulinal spore germination (there being a certain advantages over glutaraldehyde (11) in that they kill loss of heat resistance), a property that is pH dependent and spores rapidly, so that instruments could be sterilized rather appears at pH values of <6 (165, 196, 197). Potassium than disinfected between patients during busy endoscopy sorbate delays C. botulinum growth and toxin production in sessions. They can, however, cause instrument damage. The cured meats (64) and, when added to acidic foods, is activity of hypochlorites is reduced drastically in the pres- hydrolyzed to sorbic acid. The acid is effective in the ence of organic matter, whereas organic chlorine compounds undissociated form (pKa, 4.75), and the maximum pH for are less susceptible (23). Chlorine dioxide does not form activity is ca. 6 to 6.5. In addition to the effect on germina- chlorinated organic compounds and is an effective sporicide, tion noted above, sorbate delays or inhibits the outgrowth of and its activity is not significantly affected by pH (70). C. botulinum spores (20, 21). Sorbate plus nitrite is an Concentrations required and conditions of use preclude effective combination also, but the need to eliminate the the wide use of hydrogen peroxide as an effective sporicide. latter as a preservative does not make this a viable proposi- Nevertheless, peroxide has been used for sterilizing food tion. contact surfaces, that is, in obtaining commercial sterility of Antibacterial agents are also used as preservatives in the packaging material when rapid spore destruction is pharmaceutical and cosmetic products. Here, however, spe- required. Its medical (e.g., for cleansing wounds and for ear cific sporeforming agents are not necessarily the major drop formulations) and other uses do not usually rely on its problem (172). sporicidal activity. EtO is mainly used as a chemical sterilizing agent (35, CONCLUSIONS 173), but has also been used as a decontamination agent for articles handled by tuberculosis patients. In the United Comparatively few bactericidal agents are actively spori- Kingdom, it is one of the pharmacopoeial methods described cidal. The most important chemical sporicides are glutaral- for sterilizing powders, and Russell (173) has listed equip- dehyde, formaldehyde (liquid and vapor forms), chlorine- ment that has been sterilized by EtO, in each instance with releasing agents, peroxygens, and ethylene oxide. Ozone spores used as an indicator for satisfactory sterilization. may become an important addition in the near future. Even These materials included various ophthalmic instruments, so, activity against spores is invariably considerably slower anesthetic equipment, heart-lung machines, disposable sy- than against vegetative cells, and concentrations are higher ringes, and hospital blankets. A problem always associated for a sporicidal action to be achieved. Bactericidal and with EtO is the possibility of toxic effects arising from bacteriostatic chemicals that are not sporicidal are usually residual EtO present in products (162). Although EtO gas sporostatic, preventing spore germination or outgrowth or diffuses rapidly in open air, porous materials adsorb the gas both. Exact mechanisms of sporicidal activity and of spore during the sterilization cycle in various amounts and then resistance have yet to be elucidated, and further studies are require various poststerilization periods for desorption of undoubtedly necessary. residual gas. In the clinical context, a rational approach to disinfectants The antibacterial agents with sporicidal activity described and sterilization, involving a consideration of both patient in this section, therefore, can be used as chemical sterilizing risk and the treatment of equipment and environment (9, 10, agents. Two points must be added, however: (i) they are 60), is necessary. Moist heat is the preferred form of 114 RUSSELL CLIN. MICROBIOL. REV.

sterilization (usually at temperatures of 121'C or above) or, shock affects permeability and resistance of Bacillus stearo- as low-temperature steam (e.g., 730C), of disinfection. Other thermophilus spores. Appl. Environ. Microbiol. 54:2515-2520. specific methods of sterilization, such as ionizing radiation, 19 Beveridge, T. J., F. M. R. Williams, and J. J. Koral. 1975. The effect of chemical fixatives on cell walls of Bacillus subtilis. dry heat, filtration, and gaseous chemical agents (ethylene Can. J. Microbiol. 24:1439-1451. oxide and low-temperature steam with formaldehyde) are 20. Blocher, J. C., and F. F. Busta. 1983. Bacterial spore resistance used when relevant. Chemical disinfectants (liquid chemical to acid. Food Technol. 37(11):87-89. sterilants) should only be used when other methods of 21. Blocher, J. C., and F. F. Busta. 1985. Multiple modes of sterilization are inappropriate. Thus, glutaraldehyde is used inhibition of spore germination and outgrowth by reduced pH for sterilization of medical equipment when heat cannot be and sorbate. J. Appl. Bacteriol. 59:467-478. used; for example, articles in a high-risk category may be 22. Bloomfield, S. F., and G. A. Miles. 1978. The antibacterial thermolabile but require a process that is sporicidal. properties of sodium dichloroisocyanurate and sodium hy- pochlorite formulations. J. Appl. Bacteriol. 46:65-73. LITERATURE CITED 23. Bloomfield, S. F., and E. E. Uso. 1985. The antibacterial 1. Abdelaziz, A. A., and M. A. El-Nakeeb. 1988. Sporicidal properties of sodium hypochlorite and sodium dichloroisocy- activity of local anesthetics and their binary combinations with anurate as hospital disinfectants. J. Hosp. Infect. 6:20-30. preservatives. J. Clin. Pharm. Ther. 13:249-256. 24. Bohin, J.-P., and B. Lubochinsky. 1982. Alcohol-resistant 2. Ando, Y. 1973. 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The tion: water and wastewater. Ann Arbor Science Publishers influence of gramicidin S on hydrophobicity of germinating Inc., Ann Arbor, Mich. Bacillus brevis spores. Arch. Microbiol. 142:51-54. 148. Parker, M. S. 1969. Some effects of preservatives on the 170. Rubbo, S. D., J. F. Gardner, and R. L. Webb. 1967. Biocidal development of bacterial spores. J. Appl. Bacteriol. 32:322- activities of glutaraldehyde and related compounds. J. Appl. 328. Bacteriol. 30:78-87. 149. Parker, M. S., and T. J. Bradley. 1968. A reversible inhibition 171. Russell, A. D. 1971. The destruction of bacterial spores, p. of the germination of bacterial spores. Can. J. Microbiol. 451-612. In W. B. Hugo (ed.), Inhibition and destruction of the 14:745-746. microbial cell. Academic Press, Inc., New York. 150. Pepper, R. E., and V. L. Chandler. 1963. Sporicidal activity of 172. Russell, A. D. 1982. Factors influencing the efficacy of antimi- alkaline alcoholic saturated dialdehyde solutions. Appl. Micro- crobial agents, p. 107-133. In A. D. Russell, W. B. Hugo, and biol. 11:384-388. G. A. J. Ayliffe (ed.), Principles and practice of disinfection, 151. Phillips, C. R. 1949. The sterilizing action of gaseous ethylene preservation and sterilization. Blackwell Scientific Publica- oxide. II. Sterilization of contaminated objects with ethylene tions, Ltd., Oxford. oxide and related compounds: time, concentration and temper- 173. Russell, A. D. 1982. The destruction of bacterial spores. ature relationships. Am. J. Hyg. 50:280-289. Academic Press, Inc., New York. 152. Phillips, C. R., and S. Kaye. 1949. The sterilizing action of 174. Russell, A. D. 1983. Mechanisms of action of chemical spori- ethylene oxide. I. Review. Am. J. Hyg. 50:270-279. cidal and sporostatic agents. Int. J. Pharm. 16:127-140. 153. Power, E. G. M., B. N. Dancer, and A. D. Russell. 1988. 175. Russell, A. D. 1990. The effects of chemical and physical agents Emergence of resistance to glutaraldehyde in spores of Bacil- on microbes: disinfection and sterilization. In Topley & lus subtilis 168. FEMS Microbiol. Lett. 50:223-226. Wilson's principles of bacteriology, virology and immunity, 154. Power, E. G. M., B. N. Dancer, and A. D. Russell. 1989. 8th ed. Edward Arnold, London. Possible mechanisms for the revival of glutaraldehyde-treated 176. Russell, A. D.,I. Ahonkhai, and D. T. Rogers. 1979. Microbi- spores of Bacillus subtilis NCTC 8236. J. Appl. Bacteriol. ological applications of the inactivation of antibiotics and other 67:91-98. antimicrobial agents. J. Appl. Bacteriol. 46:207-245. 155. Power, E. G. M., and A. D. Russell. 1988. Assessment of 'Cold 177. Russell, A. D., B. N. Dancer, E. G. M. Power, and L. A. Sterilog Glutaraldehyde Monitor.' J. Hosp. Infect. 11:376-380. Shaker. 1989. Mechanisms of bacterial spore resistance to 156. Power, E. G. M., and A. D. Russell. 1989. Glutaraldehyde: its disinfectants, p. 9-29. In Proceedings, 4th Conference on uptake by sporing and non-sporing bacteria, rubber, plastic Progress in Chemical Disinfection. and an endoscope. J. Apple. Bacteriol. 67:329-342. 178. Russell, A. D., S. A. Hammond, and J. R. Morgan. 1986. 156a.Power, E. G. M., and A. D. Russell. 1990. Uptake of L- Bacterial resistance to antiseptics and disinfectants. J. Hosp. 14C-alanine to glutaraldehyde-treated and untreated spores of Infect. 7:213-225. Bacillus subtilis. FEMS Microbiol. Lett. 66:271-276. 179. Russell, A. D., and D. Hopwood. 1976. The biological uses and 157. Prasad, C. 1974. Initiation of spore germination in Bacillus importance of glutaraldehyde. Prog. Med. Chem. 13:271-301. subtilis: relationship to inhibitionof L-alanine metabolism. J. 180. Russell, A. D., and W. B. Hugo. 1987. Chemical disinfectants, Bacteriol. 119:805-810. p. 1242. In A. H. Linton, W. B. Hugo, and A. D. Russell 158. Quinn, P. J. 1987. 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181. Russell, A. D., B. D. Jones, and P. Milburn. 1985. Reversal of antibacterial activity, p. 213-276. In J. R. Norris and D. W. the inhibition of bacterial spore germination and outgrowth by Ribbons (ed.), Methods in microbiology, vol. 7B. Academic antibacterial agents. Int. J. Pharm. 25:105-112. Press, Inc., New York. 182. Russell, A. D., and T. J. Munton. 1974. Bactericidal and 203. Starke, R. L., D. Ferguson, P. Garza, and N. A. Miner. 1975. bacteriostatic activity of glutaraldehyde and its interaction An evaluation of the Association of Official Analytical Chem- with lysine and proteins. Microbios 11:147-152. ists sporicidal test methods. Dev. Ind. Microbiol. 16:31-36. 183. Rutala, W. A. 1987. Disinfection, sterilization and waste dis- 204. Stevenson, K. E., and B. D. Shafer. 1983. Bacterial spore posal, p. 257-282. In R. P. Wenzel (ed.), Prevention and resistance to hydrogen peroxide. Food Technol. 37(11):111- control of nosocomial infections. The Williams & Wilkins Co., 114. Baltimore. 205. Stewart, G. S. A. B., K. Johnstone, E. Hagelberg, and D. J. 184. Sammons, R. L., A. Moir, and D. A. Smith. 1981. Isolation and Ellar. 1981. Commitment of bacterial spores to germinate. A properties of spore germination mutants of Bacillus subtilis 168 measure of the trigger reaction. Biochem. J. 198:101-106. deficient in the initiation of germination. J. Gen. Microbiol. 206. Stonehill, A. A., S. Krop, and P. M. Borick. 1963. Buffered 124:229-241. glutaraldehyde, a new chemical sterilizing solution. Am. J. 185. Scott, E. M., and S. P. Gorman. 1983. Sterilization with Hosp. Pharm. 20:458-465. glutaraldehyde, p. 65-88. In S. S. Block (ed.), Disinfection, 207. Sykes, G. 1965. Disinfection and sterilization, 2nd ed. F. & N. sterilization and preservation, 3rd ed. Lea & Febiger, Phila- Spon, London. delphia. 208. Sykes, G. 1970. The sporicidal properties of chemical disinfec- 186. Scott, E. M., and E. P. Gorman. 1987. Chemical disinfectants, tants. J. Apple. Bacteriol. 33:147-156. antiseptics and preservatives, p. 226-252. In W. B. Hugo and 209. Takahashi, I., and L. W. MacKenzie. 1982. Effects of inhibitory A. D. Russell (ed.), Pharmaceutical microbiology, 4th ed. agents on sporulation of Bacillus subtilis. Can. J. Microbiol. Blackwell Scientific Publications, Ltd., Oxford. 28:80-86. 187. Setlow, P. 1988. Small, acid-soluble spore proteins of Bacillus 210. Tawasatani, T., M. Kakezawa, and I. Shibasaki. 1979. Role of species: structure, synthesis, genetics, function and degrada- cellular calcium in the variation of propylene oxide sensitivity tion. Annu. Rev. Microbiol. 42:319-338. of bacterial spores. Hakko Kyokaishi 57:203-213. (In Japa- 188. Shaker, L. A., B. N. Dancer, A. D. Russell, and J. R. Furr. nese.) 1988. Emergence and development of chlorhexidine resistance 211. Thomas, S. 1977. Effect of high concentrations of glutaralde- during sporulation of Bacillus subtilis 168. FEMS Microbiol. hyde upon bacterial spores. Microbios Lett. 4:199-204. Lett. 51:73-76. 212. Thomas, S., and A. D. Russell. 1974. Studies on the mechanism 189. Shaker, L. A., J. R. Furr, and A. D. Russell. 1988. Mechanism of the sporicidal action of glutaraldehyde. J. Apple. Bacteriol. of resistance of Bacillus subtilis spores to chlorhexidine. J. 37:83-92. Appl. Bacteriol. 64:531-539. 213. Thomas, S., and A. D. Russell. 1974. Temperature-induced 190. Shaker, L. A., A. D. Russell, and J. R. Furr. 1986. Aspects of changes in the sporicidal activity and chemical properties of the action of chlorhexidine on bacterial spores. Int. J. Pharm. glutaraldehyde. Apple. Microbiol. 28:331-335. 34:51-56. 214. Thomas, S., and A. D. Russell. 1975. Sensitivity and resistance 191. Shibata, H., M. Uchida, H. Hayashi, and I. Tani. 1979. Effect of to glutaraldehyde of the hydrogen and calcium forms of Bacil- trichloracetic acid treatment on certain properties of spores of lus subtilis spores. J. Apple. Bacteriol. 38:315-317. Bacillus cereus T. Microbiol. Immunol. 23:339-347. 215. Toledo, P. T., S. E. Escher, and J. C. Ayres. 1973. Sporicidal 192. Sierra, G., and R. M. G. Boucher. 1971. Ultrasonic synergistic properties of hydrogen peroxide against food-spoilage organ- effects in liquid-phase chemical sterilization. Apple. Microbiol. isms. Apple. Microbiol. 26:592-597. 22:160-164. 216. Tolon, M., E. Thofern, and S. E. Miederer. 1976. Disinfection 193. Smoot, L. A., and M. D. Pierson. 1982. Inhibition and control procedures of fiberscopes in endoscopy departments. Endos- of bacterial spore germination. J. Food Prot. 45:84-92. copy 8:24-29. 194. Snyder, R. W., and E. L. Cheatle. 1965. Alkaline glutaralde- 217. Treadwell, P. E., G. J. Jann, and A. J. Salle. 1958. Studies on hyde-an effective disinfectant. Am. J. Hosp. Pharm. 22: factors affecting the rapid germination of spores of Clostridium 321-327. botulinum. J. Bacteriol. 76:549-555. 195. Sofos, J. N., and F. F. Busta. 1981. Antimicrobial activity of 218. Trueman, J. 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by heat and chemical agents. J. Apple. Biochem. 1:71-76. 235. Whipple, E. B., and M. Ruta. 1974. Structure of aqueous 227. Waites, W. M., and C. E. Bayliss. 1980. Microbial growth and glutaraldehyde. J. Org. Chem. 39:1666-1668. survival in extremes of environment. Soc. Apple. Bacteriol. 236. Woods, D. R., and D. T. Jones. 1986. Physiological responses Tech. Ser. 15:159-172. of Bacteroides and Clostridium strains to environmental stress 228. Waites, W. M., and C. E. Bayliss. 1984. The revival of injured factors. Adv. Microb. Physiol. 28:1-64. microbes. Soc. Apple. Bacteriol. Symp. Ser. 12:221-240. 237. Wyatt, L. R., and W. M. Waites. 1975. The effect ofchlorine on 229. Waites, W. M., C. E. Bayliss, N. R. King, and A. M. C. Davies. spores of Clostridium bifermentans, Bacillus subtilis and Ba- 1979. The effect of transition metal ions on the resistance of cillus cereus. J. Gen. Microbiol. 89:327-334. bacterial spores to hydrogen peroxide and to heat. J. Gen. 238. Yasuda, Y., and K. Tochikubo. 1984. Relation between D- Microbiol. 112:225-233. glucose and L- and D-alanine in the initiation of germination of 230. Waites, W. M., S. E. Harding, D. R. Fowler, S. H. Jones, D. Bacillus subtilis spore. Microbiol. Immunol. 28:197-207. Shaw, and M. Martin. 1988. The destruction of spores of 239. Yasuda, Y., and K. Tochikubo. 1985. Germination-initiation Bacillus subtilis by the combined effects of hydrogen perioxide and inhibitory activities of L- and D-alanine analogues for B. and ultraviolet light. Lett. Apple. Microbiol. 7:139-140. subtilis spores. Modification of methyl group of L- and D- 231. Waites, W. M., L. R. Wyatt, N. R. King, and C. E. Bayliss. alanine. Microbiol. Immunol. 29:229-241. 1976. Changes in spores of Clostridium bifermentans caused 240. Yasuda, Y., and K. Tochikubo. 1985. Disappearance of the by treatment with hydrogen peroxide and cations. J. Gen. cooperative effect of glucose on L-alanine binding during heat Microbiol. 93:388-396. activation ofgermination ofBacillus subtilis spores. Microbiol. 232. WallhAuser, K. H. 1984. Antimicrobial preservatives used by Immunol. 29:1011-1017. the cosmetic industry, p. 605-745. In J. J. Kabara (ed.), 241. Yasuda, Y., K. Tochikubo, Y. Hachisuka, H. Tomida, and K. Cosmetic and drug preservation: principles and practice. Mar- Ikeda. 1982. Quantitative structure-inhibitory activity relation- cel Dekker, Inc., New York. ships of phenols and fatty acids for Bacillus subtilis spore 233. Warth, A. D. 1978. Molecular structure of the bacterial spore. germination. J. Med. Chem. 25:315-320. Adv. Microb. Physiol. 17:1-45. 242. Yasuda-Yasaki, K., S. Namiki-Kanie, and Y. Hachisuka. 1978. 234. Watanabe, K., and S. Takesue. 1976. Selective inhibition of the Inhibition of Bacillus subtilis spore germination by various germination of Bacillus megaterium spores by alkyl p-hy- hydrophobic compounds: demonstration of hydrophobic char- droxybenzoates. Chem. Pharm. Bull. 24:224-229. acter of the L-alanine receptor site. J. Bacteriol. 136:484-490.