Transcript
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CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 99-119 Vol. 3, No. 20893-8512/90/020099-21$02.00/0Copyright © 1990, American Society for Microbiology

Bacterial Spores and Chemical Sporicidal AgentsA. D. RUSSELL

Welsh School of Pharmacy, University of Wales College of Cardiff, Cardiff, CFJ 3XF, Wales

INTRODUCTION................................................. 99THE BACTERIAL SPORE ................................................. 99SPOROSTATIC AND SPORICIDAL ACTIVITY .................................................. 100Group A: Sporostatic Compounds ................................................. 101

Phenols and cresols................................................. 101Organic acids and esters ................................................. 101Alcohols ................................................. 101QACs ................................................. 101Biguanides ................................................. 101Organomercury compounds................................................. 101

Group B: Sporicidal Compounds ................................................. 102Glutaraldehyde ................................................. 102Formaldehyde ................................................. 102Other aldehydes.................................................. 103Chlorine-releasing agents .................................................. 103Iodine and iodophors .................................................. 103Peroxygens .................................................. 103Ethylene oxide ................................................. 104P-Propiolactone ................................................. 104Other gases................................................. 105

RECOVERY AND REVIVAL OF INJURED SPORES ................................................. 105SPOROGENESIS, SUSCEPTIBILITY, AND RESISTANCE .................................................. 106

Sporulation ................................................. 107Germination................................................. 108Outgrowth ................................................. 110

OVERCOMING SPORE RESISTANCE ................................................. 110MECHANISMS OF SPORICIDAL ACTION..................................................111MEDICAL AND OTHER USES OF CHEMICAL SPORICIDES ................................................. 112

Sporicidal Agents ................................................. 112Inhibitors of Germination and Outgrowth ................................................. 113

CONCLUSIONS ................................................. 113LITERATURE CITED................................................. 114

INTRODUCTION

Bacterial spores are highly resistant to chemical andphysical agents (25, 88, 89, 91, 102, 139, 158, 166, 171-174,178, 207, 208, 224, 225). Processes designed to achievesterilization of food, pharmaceutical, medical, and otherproducts have thus, of necessity, had to take this high levelof resistance into account. Spores are also of importance inother contexts, notably, (i) as food-poisoning agents (Clos-tridium botulinum, C. perfringens, and Bacillus cereus), (ii)as etiological agents (C. perfringens and C. tetani) in someinfections, and (iii) as sources of antibiotics, toxins, andinsecticides. Add to these the complex and fascinating seriesof events that take place during sporulation, germination,and outgrowth and the stage is set for a comprehensive studyencompassing many scientific and medical disciplines, sev-eral of which are outside the scope of the present paper.

This paper will deal with chemical sporicidal agents of thedisinfectant type. Such chemicals are comparatively few innumber and their activity is often susceptible to environmen-tal conditions, at least some of which can be readily con-trolled. Other agents that are bactericidal and sporostatic butnot usually sporicidal will also be considered when relevant.More effective sporicidal action will only be achieved by

learning more about the ways in which sporicides act orspores resist their action, and due attention will be paid tothese aspects. Finally, the clinical uses of sporicidal agentswill be discussed.

General aspects of disinfection and disinfectants are to befound in references 73, 101, and 183. These include details,when relevant, of sporicidal activity. Spore resistance isdescribed by Russell et al. (177) and Gardner and Peel (72).

In the United States, commercially available disinfectantsare regulated by the Environmental Protection Agency andmust be used according to the directions specified on theirlabels. Workers elsewhere should be familiar with regula-tions pertaining to their own country.

THE BACTERIAL SPORE

The most important sporeformers are members of thegenera Bacillus and Clostridium. Certain other bacteria,e.g., Sporosarcinae, Desulfomaculum, and Sporolactobacil-lus spp. (52), can also form spores, but will not be consideredhere. True endospores are also produced by thermophilicactinomycetes. Thermoactinomyces vulgaris spores are

highly refractile, do not take up simple stains, have a typical

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CLIN. MICROBIOL. REV.

CX

FIG. 1. "Typical" bacterial spore. The exosporium is present insome, but not all, types of spores. EXO, Exosporium; OSC, outerspore coat; ISC, inner spore coat; CX, cortex-; GCW, germ cell wall;PM, plasma membrane.

spore structure, contain dipicolinic acid, and are heat resis-tant (173).The structure of a so-called typical bacterial spore is

depicted in Fig. 1. It is clear that the spore is a complexentity, being composed of several different layers, some ofwhich are implicated in their greater resistance than vegeta-tive cells to chemical or physical processes. The molecularstructure of the bacterial spore is considered in detail byEllar (57) and Warth (233). The germ cell (protoplast or core)and germ cell wall are surrounded by the cortex, external towhich are the inner and denser outer spore coats. Anexosporium is present in some spores, but may surround justone dense spore coat.

In terms of its macromolecular constituents (Table 1), theprotoplast is the location of RNA, DNA, dipicolinic acid,and most of the calcium, potassium, manganese, and phos-phorus present in the spore. Also present is a substantialamount of low-molecular-weight basic proteins which arerapidly degraded during germination (187).The cortex consists largely of peptidoglycan, some 45 to

60% of the muramic acid residues not having either a peptideor an N-acetyl substituent but instead forming an internalamide, muramic lactam (233). Peptidoglycan is the site ofaction of lysozyme and of nitrous acid. A dense inner layer

TABLE 1. Chemical composition of bacterial spores

component Composition Comment

Outer spore Mainly protein Alkali resistant; removedcoat by disulfide bond-re-

ducing agentsInner spore Mainly protein Alkali soluble

coatCortex Mainly peptidoglycan Differs from peptidogly-

can of vegetative cellwall

Core Protein, DNA, RNA, Unique spore proteinsDPA,a divalent cat- associated with DNAions

a DPA, Dipicolinic acid.

TABLE 2. Agents with bactericidal, sporostatic,and sporicidal activity

Bactericidal agents Bactericidal agents Commentthat are sporostatic that are sporicidal

Group APhenols None in group A Even high concentra-Organic acids and tions for prolonged

estersa periods at ambientQACs temp are not spori-Biguanides cidal; may beOrganomercurials sporicidal at ele-Alcohols vated temperatures

Group BGlutaraldehyde All in group B Low concentrationsFormaldehyde are sporostatic;Iodine compounds usually much

higher concentra-Chlorine compounds tioner neededtons are neededHydrogen peroxide for sporicidal effectPeroxy acidsEthylene oxide13-Propiolactonea For example, the parabens [methyl, ethyl, propyl, and butyl esters of

para-(4)-hydroxybenzoic acid].

(cortical membrane, germ cell wall, primordial cell wall) ofthe cortex develops into the cell wall of the emergent cellwhen the cortex is degraded during germination.Two membranes, the inner and outer forespore mem-

branes, surround the forespore during germination. Theinner forespore membrane eventually becomes the cytoplas-mic membrane of the germinating spore, whereas the outerforespore membrane persists in the spore integuments.The spore coats make up a major portion of the spore

(139), consisting mainly of protein with smaller amounts ofcomplex carbohydrates and lipid and possibly large amountsof phosphorus. The outer spore coat contains the alkali-resistant protein fraction and is associated with the presenceof disulfide-rich bonds. The alkali-soluble fraction is found inthe inner spore coats and consists predominantly of acidicpolypeptides which can be dissociated to their unit compo-nents by treatment with sodium dodecyl sulfate.From this brief consideration of the structure and compo-

sition of the bacterial spore, it is obvious that several sitesexist for attack by biocides and equally obvious that thespore can possess barriers which limit biocide penetration. Itis the purpose of this review not only to describe the activity,properties, and uses of sporicidal agents but also to considertheir mechanism of action, insofar as this is known, howresistance may be presented by the spore, and how this maybe overcome.

SPOROSTATIC AND SPORICIDAL ACTIVITY

Comparatively few antibacterial agents are actively spori-cidal (101, 173, 180). Even quite powerful bactericides maybe inhibitory to spore germination or outgrowth or both, i.e.,sporostatic, rather than sporicidal. Examples include phe-nols and cresols, quaternary ammonium compounds(QACs), biguanides such as chlorhexidine, organic mercurycompounds, and alcohols (group A in Table 2). Sporicidalactivity may, however, be achieved at elevated tempera-tures. It is clear from Table 3 that concentrations effectingsporostasis are usually very close to those that inhibitvegetative cell growth.

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SPORICIDAL AGENTS 101

TABLE 3. Comparison of bacteriostatic andsporostatic concentrations

Antibacterial Bacteriostatic concn Sporostatic concnagent (%, wt/vol) (%, wt vol)

Benzalkonium chloride 0.0004-0.0016 0.0005Cetylpyridinium chlo- 0.0005 0.00025

rideChlorhexidine diace- 0.0001 0.0001

tate or gluconateChlorocresol 0.02 0.02Cresol 0.08 0.1Phenol 0.2 0.2Phenylmercuric nitrate 0.00001-0.0001 0.00002

or acetatea Based on reference 173.

It is also apparent from Table 4 that even chemicals thatare considered to be sporicidal require much higher concen-trations for this effect than for bactericidal activity. Also, atime factor must be considered since spores must invariablybe exposed for longer periods. A recent example of this isdescribed by Power and Russell (156), who demonstrate that2% alkaline glutaraldehyde will sterilize an inoculum of ca.108 CFU of Escherichia coli, Staphylococcus aureus, andvegetative cells of B. subtilis per ml within 10 min at 22C,whereas B. subtilis spores require several hours.Agents that are actively sporicidal (group B in Table 2)

include aldehydes, halogens, peroxygens, and gaseous orvapor-phase disinfectants. The properties of chemical com-pounds in both groups A and B are considered below.

Group A: Sporostatic Compounds

Chemical compounds in group A are not sporicidal butinhibit germination or outgrowth at concentrations similar tobacteriostatic ones.

Phenols and cresols. Even at high concentrations, phenolicdisinfectants are poorly sporicidal (102, 180), 2.5 and 5%(wt/vol) having little effect on B. subtilis spores even after100 h at 25 or 37TC. In contrast, concentrations as low as0.2% (phenol), 0.08% (cresol), and 0.02% (chlorocresol)(wt/vol) are all effective inhibitors of germination (173).Furthermore, sporicidal activity is greatly accelerated whenphenolics are used at elevated temperatures (33). Such aprocess, utilizing 0.2% (wt/vol) chlorocresol at 98 to 100'C,was for many years a pharmacopoeial process in the UnitedKingdom for sterilizing certain injectable and ophthalmicproducts, but is no longer official.

TABLE 4. Comparison of bactericidal andsporicidal concentrations

Antibacterial Bactericidal concn Sporicidal concnagent (%, wt/vol) (%, wt/vol)

Chlorocresol 0.1 >0.4Cresol 0.3 >0.5Phenol 0.5 >5.0Phenylmercuric nitrate 0.002 >0.02Chlorhexidine diacetate 0.002 >0.05Cetylpyridinium chloride 0.002 >0.05bGlutaraldehyde <0.1 2.0Formaldehyde <1 4-8Hypochlorite 1-2 ppm 20 ppm

a Kill may depend on pH and temperature and on period of treatment.b Not sporicidal at this concentration at ambient temperatures.

Organic acids and esters. Organic acids such as benzoicand sorbic acids and esters (parabens) of para-(4)-hydroxy-benzoic acid are widely used as preservatives (102, 126, 180,196, 232). They are bactericidal but not sporicidal. Theparabens inhibit the growth and toxin production of C.botulinum (165) and act at the germination stage. Theiractivity is only slightly affected over the pH range 4 to 8,whereas organic acids are most active in the undissociatedform, at low pH values.

Alcohols. Ethanol is rapidly lethal to nonsporing bacteriawhen the alcohol is used at appropriate concentrations, buthas no sporicidal activity (171, 173). The addition of 1%sodium or potassium hydroxide, various acids, or 10% amyl-m-cresol to 70% alcohol is claimed to enhance sporicidalactivity (208). Initial sporicidal activity of an ethanol-hy-pochlorite mixture is high but decreases on storage (40).

Other alcohols, namely, methanol (methyl alcohol), pro-pan-1-ol, propan-2-ol (isopropyl alcohol, isopropanol), phen-ethyl alcohol (phenylethanol), and octanol, also lack spori-cidal activity (102, 180). It is of interest, however, to notethat fresh mixtures of methanol (15%) and hypochlorite havea low sporicidal activity and that this activity increases asthe mixture ages, in contrast to ethanol-hypochlorite orpropan-2-ol plus hypochlorite, when the reverse applies (40,51). Furthermore, increasing the methanol concentration to25%, and especially to 50%, produces a rapid initial spori-cidal action which can be maintained for at least 8 h afterpreparation (51). Alcohols have been used for the selectiveisolation of sporeforming bacteria (122).QACs. The QACs can be considered derivatives of ammo-

nium salts (NH4X) in which the hydrogen atoms arereplaced by alkyl groups (R1 to R4). The sum of the carbonatoms in the four R groups is >10, and at least one of the Rgroups must have a chain length in the range C8 to C18.As a group, the QACs are bactericidal in low concentra-

tions to nonmycobacterial, nonsporeforming, gram-positivebacteria, are less active against gram-negative bacteria, andare not sporicidal (41, 181). Low concentrations are, how-ever, sporostatic (Table 3), the QACs inhibiting outgrowthbut not germination (172, 173). Activity is markedly reducedin the presence of organic matter and is greater at alkalinethan at acid pH.

Biguanides. The most important biguanide is chlorhexi-dine, which is used as the acetate (diacetate) and gluconatesalts. It is an effective bacteriostatic and bactericidal agenttowards many gram-positive and gram-negative bacteria, butis not mycobactericidal and is sporostatic rather than spori-cidal (172, 173). Chlorhexidine is sporicidal at elevatedtemperatures (77, 190) and, like the QACs, it inhibits out-growth rather than germination (189). Activity is greatlyreduced in the presence of organic matter and is greater atalkaline than at acid pH.Organomercury compounds. In the last century, it was

claimed that the inorganic mercury compound mercuricchloride was rapidly sporicidal towards B. anthracis. It wassubsequently demonstrated that this incorrect conclusionwas based on a failure to control adequately sporostasis inthe subculture medium (reviewed in reference 174). Organo-mercury compounds such as phenylmercuric nitrate, phe-nylmercuric acid, and thiomersal (merthiolate) are importantpreservatives in many types of pharmaceutical products.These compounds are bacteriostatic and bactericidal and are

effective sporostatic agents at low concentrations, but are

only sporicidal when used at high temperatures (171, 173).

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Group B: Sporicidal Compounds

Chemical agents in group B are sporostatic at low concen-trations and sporicidal at much higher levels.

Glutaraldehyde. Glutaraldehyde [pentanedial; CHO(CH2)3 CHO] is a powerful antimicrobial agent and elec-tron microscope fixative (84, 185). Its activity depends on

pH, alkaline solutions being considerably more effectivethan acid ones (84, 185, 186). There is, however, a complexrelationship among the parameters of concentration, temper-ature, and pH. The rate of bactericidal and sporicidal activ-ity for aqueous acid solution is considerably lower than thatfor activated alkaline solution (26, 27, 84, 120, 137, 212). Astemperature is increased, however, this difference betweenalkaline and acid solutions is reduced (84, 192, 213).One of the earliest indications of antimicrobial activity of

glutaraldehyde arose from a survey of the sporicidal activityof saturated dialdehydes in a search for an efficient substitutefor formaldehyde. This search revealed (150) that glutaral-dehyde in alcoholic solution was superior as a sporicidalagent to both formaldehyde and glyoxal. Stonehill et al. (206)and Snyder and Cheatle (194) demonstrated that aqueoussolutions of the dialdehyde were acidic and needed to bebuffered ("activated") by suitable alkalinating agents to a

pH of 7.5 to 8.5 for antimicrobial activity. A 2% (wt/vol)glutaraldehyde solution activated with 0.3% (wt/vol) sodiumbicarbonate was advocated to provide the minimum concen-tration and conditions necessary for rapid sporicidal activity.At this in-use concentration, the dialdehyde was capable ofkilling spores of Bacillus and Clostridium spp. in 3 h (26,203). Rubbo et al. (170) reported a 4-log (99.9%) kill of sporesof B. anthracis and C. tetani in 15 and 30 min, respectively.Not all species are equally susceptible to glutaraldehyde,and B. subtilis (29) and B. pumilus (170) appear to be themost resistant. With B. subtilis spores in liquid suspension,a 3-h contact period with 2% alkaline glutaraldehyde pro-duces ca. a 6-log drop in viable count (73, 116, 131, 192).Using the Association of Official Analytical Chemists spori-cidal test (8) and vacuum-dried spores of B. subtilis, how-ever, Boucher (29) found that 10 h was necessary to achievea complete kill. Obviously, this long period cannot always beused in practice. The possible revival of glutaraldehyde-treated spores will be considered later.

Vegetative bacteria are more susceptible to the action ofglutaraldehyde, a concentration as low as 0.02% alkalinealdehyde achieving an inactivation factor of 104 to 106 within20 min at 20°C (164).At alkaline pH, glutaraldehyde solutions have a tendency

to polymerize; polymerization in acid solution is very slow.Consequently, since monomeric glutaraldehyde is consid-ered to be the active moiety (170) with interaction betweenamino groups in protein enhanced at alkaline pH (179), it isclear that acid solutions are more stable but less active andalkaline solutions are less stable but more active. Theseproblems of stability and active life have prompted thedevelopment of novel formulations to overcome these draw-backs in use. Alkalination of glutaraldehyde produces agradual decrease in aldehyde concentration (25, 155), the fallbeing temperature dependent (213). Some formulations uti-lize the benefits conferred by formulating in the loweralkaline range of ca. pH 7.5. One such product, a stabilizedglutaraldehyde solution (131), also contains surfactants topromote rinsing of surfaces and is claimed to have the usualantimicrobial activity while also maintaining a stable glutar-aldehyde concentration and pH over 28 days. In manyinstances, novel formulations have been produced which are

based on acid rather than alkaline glutaraldehyde, therebybenefiting from the stability inherent in such solutions. Theimproved sporicidal activity claimed for these acidic solu-tions has often been obtained by the addition of agents thatproduce a potentiated or synergistic effect with the dialde-hyde, e.g., nonionic surfactants (28-31) and anionic surfac-tants (81). Inorganic cation-anionic surfactant combinationsgreatly increase the antimicrobial activity at acid pH with afurther increase in efficiency at higher temperatures (81).These solutions have a shelf life of years in terms ofglutaraldehyde concentration, polymerization, and pH.Babb et al. (11) have examined different glutaraldehydeformulations and showed that acid dialdehyde preparations,although more stable than the alkaline ones, were lesssporicidal and more corrosive.An enhanced activity is claimed for a combination (Spori-

cidin; The Sporicidin Co., Washington, D.C.) of glutaralde-hyde with sodium phenate and phenol (106, 107). Studiesfrom our laboratory (E. G. M. Power and A. D. Russell, J.Appl. Bacteriol., in press) agree to claims that the undilutedmixture is sporicidal but not that a 1:16 dilution is sporicidal.Inadequate neutralization of the high phenate concentrationmay be a contributory factor in reaching an erroneousconclusion.Formaldehyde. Formaldehyde (methanal, HCHO) is used

in both the gaseous and liquid forms (171-173). Formalde-hyde solution (Formalin) is an aqueous solution containingca. 34 to 38% (wt/wt) CH2O and methanol to delay polymer-ization. Formaldehyde is bactericidal and sporicidal, but at aslower rate than glutaraldehyde (170). It combines readilywith proteins and is less effective in the presence of organicmatter.

Ortenzio et al. (146) claimed that formaldehyde solutionwas rapidly sporicidal to B. subtilis but not to C. sporogenes,which was not killed after 2 h of exposure. Borax-Formalinand formaldehyde-alcohol have been found to destroy B.anthracis, C. perfringens, and C. tetani (199), although somedoubt must remain about the validity of these results sincethere could have been a failure to neutralize formaldehyde insubculture media. A lack of sporicidal activity of 8% form-aldehyde has been noted by Pepper and Chandler (150), andthis finding might take on an added significance when linkedto proposals that spores apparently inactivated by formalde-hyde may be revived by appropriate posttreatment proce-dures (200, 201). This aspect is considered in more detaillater.

Irrespective of whether aqueous or alcoholic solutions offormaldehyde are used, time-survivor curves of treatedbacterial spores often show an initial shoulder (170), al-though this is by no means a universal finding (219). Accord-ing to some workers (170), various alcohols (methanol,ethanol, and propan-2-ol) reduce the sporicidal activity offormaldehyde. The sporicidal activity of formaldehyde isinfluenced markedly by temperature, with extensive sporeinactivation at temperatures of 40°C and above (219).Formaldehyde vapor may be obtained in various ways: (i)

by evaporating appropriate dilutions of standardized batchesof commercial Formalin (containing 10% methanol); (ii) byheating paraformaldehyde or the formaldehyde polymers,urea formaldehyde and melamine formaldehyde, under con-trolled conditions of time and temperature (221). Bacterialspores and nonsporulating bacteria are fairly readily killedby formaldehyde gas (207). A linear relationship existsbetween the formaldehyde concentration and killing rate.Nordgren (144) observed that the rate of disinfection ofspores exposed to formaldehyde vapor increased as the

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SPORICIDAL AGENTS 103

TABLE 5. Comparative sporicidal activities of some aldehydesa

Aldehyde Chemical formula Sporicidal activity

Formaldehyde H * CHO FairGlyoxal CHO * CHO Only at 10% (not 2%)Malonaldehyde CHO CH2 CHO SlightSuccinaldehydec CHO (CH2)2 CHO SlightGlutaraldehyde CHO * (CH2)3 * CHO HighAdipaldehyde CHO (CH2)4 CHO Slight

a Comments on sporicidal activity of aldehydes based in part on Boucher(28-30).h Some aldehydes have been reexamined against B. subtilis NCTC 8236spores (Power and Russell, in press).

c Gigasept contains both succinaldehyde and formaldehyde and is moreactive (as a 10%o solution) than formaldehyde alone.

temperature increased, that organic matter such as blood,sputum, or soil reduced the rate of spore inactivation, andthat an increase in rate of kill occurred as the relativehumidity was raised to 50% with little increase above thisvalue. There is no universal agreement about the effect ofrelative humidity on activity. Russell (171, 172) reviewed theactivity of formaldehyde and described work claiming thatno bactericidal effect occurs unless the humidity is 70% orabove. Confusingly, Hoffman (99) has reported that thealdehyde is quite effective even at humidity values of <50%and that formaldehyde generated from paraformaldehyde ismore active than an equivalent amount generated fromFormalin solution. Low concentrations of formaldehyde aresporostatic.Other aldehydes. The sporicidal activity of aldehydes

other than glutaraldehyde and formaldehyde is equivocal(Table 5). Borick et al. (26) stated that glyoxal was spori-cidal, and Boucher (28, 30) has considered the efficacy ofvarious aldehydes. We have recently reexamined the effectof some aldehydes on B. subtilis NCTC 8236 spores (Powerand Russell, in press). Over a 5-h period at 22°C, 10%butyraldehyde had no sporicidal action against the spores(109 CFU/ml at zero time), 10% glyoxal effected a 3-logreduction, and 8% formaldehyde produced a 4-log reduction.Gigasept (Sterling-Winthrop, Surrey, United Kingdom)(containing succinaldehyde [butan-1,4-dial], formaldehyde,and 2,5-dimethoxytetrahydrofuran, pH 6.5) achieved, at 5%,a 2-log reduction and, at 10%, a 5-log reduction. Malonalde-hyde was inactive, and the greatest rates of kill wereobtained with 2% alkaline glutaraldehyde, activated Cidex(Surgikos, Arlington, Tex.) and undiluted Sporicidin (glutar-aldehyde, 2%; phenol, 7%; sodium, phenate, 1.2%; pH 7.4),all of which produced reductions of at least 8 logs. Sporicidinwas marginally the most active.

Chlorine-releasing agents. Essentially, chlorine com-pounds can be considered as being of three types: chlorinegas, which is too hazardous for normal use; sodium andcalcium hypochlorites; and chlorine-releasing agents (55,218). Of these, the agents of choice are sodium hypochlorite,which contains up to 15% (wt/vol) available chlorine, andsodium dichloroisocyanusate, which slowly releases hy-pochlorous acid (HOCl). The active species is undissociatedhypochlorous acid, the hypochlorite ion (OCl-) being con-siderably less so, and disinfection by chlorination is optimalat around pH 6, at which dissociation of HOCI is minimal.

Chlorine compounds are bactericidal and sporicidal, al-though spores are more resistant than vegetative cells (22,23, 42, 55, 56, 147, 218). Activity of the hypochlorites isgreatly reduced in the presence of organic matter. OrganicN-chloro compounds, containing the =N-Cl- group, hy-

drolyze in water to produce an amino (=NH) group; theirsporicidal activity is slower than that of the hypochlorites.Like the hypochlorites, activity of these compounds isgreater at acid than at alkaline pH. Cousins and Allan (42)have demonstrated that sodium hypochlorite was the mosteffective of five halogens against B. cereus and that B.subtilis spores were more resistant to all sporicides tested.Generally, spores of Clostridium spp. are more susceptibleto chlorine than are Bacillus spores (56).

Freshly prepared hypochlorite solutions, buffered to aboutpH 7.6, have a very rapid sporicidal activity (11, 116).Mixtures of 1.5 to 4% sodium hydroxide with sodium hy-pochlorite (200 ppm [200 pg/g] available chlorine) are muchmore rapidly sporicidal than either sodium hydroxide orhypochlorite used singly (42). This could result from aneffect of the alkali on the spore coat, thereby increasinghypochlorite penetration. Potentiation of sporicidal activityof hypochlorites is attained in the presence of methanol andother alcohols (40, 51, 116), and buffering to pH 7.6 to 8.1 ofalcohol-hypochlorite solutions produces powerful sporicidalactivity with optimum stability (51). Such solutions are still,however, inactivated by organic matter.

Iodine and iodophors. Iodine and iodophors (iodophores;literally, iodine carriers) are considered to be effectivebactericidal and sporicidal agents (207, 218). Iodine itself issparingly soluble in cold, but more soluble in hot, water.Stronger solutions can be made in potassium iodide or inalcohol. Iodine is less reactive chemically than chlorine andis less affected by the presence of organic matter; neverthe-less, these effects depend on iodine concentration. Theactivity of low, but not of high, concentrations of iodine issignificantly reduced. The sporicidal efficacy of iodine is alsopH dependent; at neutral and acid pH, diatomic iodine (12) ishighly active and hypoiodous acid (HOI) also makes somecontribution. At alkaline pH, activity is reduced, resultingfrom the formation of the hypoiodide (OF) ion, which hasonly a slight activity, and the inactive iodate (103), iodide(I-), and triiodide (13) ions. A major problem with iodine isthat it is toxic and also stains fabric and tissues.The iodophors consist of a loose complex of elemental

iodine solubilized by means of appropriate carriers whichincrease solubility while at the same time providing a sus-tained-release reservoir of iodine (85) and stabilized withphosphoric acid. Suitable carriers consist of neutral poly-mers such as nonionic surfactants and povidone (polyvi-nylpyrrolidone) and polyethylene glycols, which exhibitsurface-active properties and which, therefore, improvewetting properties, thereby aiding in penetration into organicsoil. The iodophors have been reviewed by several authors(85, 171-173, 207, 218). The concentration of free iodine inan iodophor is responsible for its bactericidal activity. Inmany iodophor preparations, the carrier is a nonionic sur-factant in which the iodine is present as micellar aggregates.When the iodophor is diluted with water, the micellesdisperse and most of the iodine is slowly liberated. Dilutionbelow the critical micelle concentration of the surfactantresults in iodine being in simple aqueous solution. Favero(60) has made the important point that iodophors formulatedas antiseptics contain much less free iodine than thoseformulated as disinfectants, which should contain 30 to 50mg of free iodine, or 70 to 150 mg of available iodine per liter.Iodophors at high concentrations may be sporicidal over a

wide pH range, but are much less potent than glutaralde-hyde; they do not stain and are nontoxic.

Peroxygens. The two important peroxygens are hydrogenperoxide (H202) and peracetic acid (CH3COOOH).

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The bactericidal and sporicidal properties of hydrogenperoxide have long been known (4-6, 16, 17, 121, 215,224-226, 229-231) and are influenced by a variety of factors.Of these, one of the most important is concentration, a lowconcentration (6%, wt/vol) being bactericidal, but onlyslowly sporicidal. However, at 25TC and levels of between 10and 20% (wt/vol), the concentration exponent is about 1.5(173). Survivor curves of spores exposed to low peroxideconcentrations frequently exhibit a distinct shoulder. A"tailing" has also been observed (36), which has beenattributed to the formation of spore clumps during treatmentand the associated spore catalase, thereby destroying hydro-gen peroxide in the immediate vicinity. Temperature exertsa marked effect on sporicidal activity; at ambient tempera-tures, peroxide is only slowly sporicidal, but the temperaturecoefficient (Q10) for each 10'C rise is about 2.5 (173). A factorthat can influence activity of peroxide is its stability; it tendsto be unstable and its decomposition is increased by metals,metallic salts, light, heat, and agitation, but it is compara-tively stable in the presence of a slight excess of acid.Decomposition can be reduced by appropriate storage.

Peracetic acid is a bactericidal and sporicidal agent (13, 14,224). It decomposes ultimately to hydrogen peroxide, aceticacid, and oxygen, which at recommended in-use concentra-tions is toxologically safe. It is considered (13, 14) to be amore potent sporicide than hydrogen peroxide, and itsactivity is reduced only slightly in the presence of organicmatter and is unaffected by the presence of catalase. Perace-tic acid is more active at pH 5 than at neutral pH.

Ethylene oxide. Ethylene oxide [EtO; (CH2)20] existsusually as a gas that is soluble in water, oils, rubber, andmost organic solvents. A major problem with its use is thatit is inflammable when in contact with air, but in practice thiscan be overcome by using mixtures of EtO with carbondioxide or fluorocarbon compounds. EtO is freely diffusibleand penetrates paper, cellophane, cardboard, fabrics, andsome plastics but less readily through polyethylene. It isunable to penetrate crystalline materials.

Early work (113-115, 151, 152) considered the chemical,physical, and microbiocidal properties of EtO. Later studieshave fully supported these findings (34, 35, 39, 58, 59, 75, 99,117, 129, 159, 162, 171-173, 180) and have shown that theactivity of EtO depends on several factors.

(i) Activity is concentration and time dependent. As wouldbe expected, the higher the concentration of EtO, the morerapid its sporicidal activity. For example, Phillips (151)calculated values of 1/k (equivalent to the time in hours at250C required [t90%I to kill 90% of B. subtilis var. globigiispores dried on cloth): values of 1/k were 7.2, 3.3, 1.6, 0.5,and 0.35 h for EtO concentrations of 22, 44, 88, 442, and 884mg/liter, respectively. As the concentration of EtO in-creases, obviously 1/k or t90% decreases.These values demonstrate clearly that EtO is only slowly

bactericidal, even a high concentration (884 mg/liter), taking0.35 h to reduce the number of viable spores by 90%, i.e., 1log cycle. This slow rate of kill is an obvious disadvantage ofthe gas and is a property that is taken into account inproviding suitable conditions for sterilization.

(ii) Activity is temperature dependent. Sporicidal activityof EtO is increased as the temperature is raised. Phillips(151) calculated that the temperature coefficient (010 or Q10)was 2.74 for each 10°C rise in temperature. The relationshipamong concentration, time, and temperature is, however,more complex than might be implied from this simplestatement. Ernst (58, 59) has shown that the death rate islogarithmic and that the Q10 of 2.74 describes the tempera-

ture effect for EtO concentrations of <880 mg/liter at tem-peratures of <350C. However, a critical temperature isreached for a particular concentration, after which an in-crease in concentration has no additional effect on the rate ofkill of bacterial spores. At higher concentrations and tem-peratures about 32TC, the kinetics become zero order withrespect to concentration, with a Q10 value of 1.9.

(iii) Activity is water vapor dependent. Of all factorsinfluencing EtO activity, moisture vapor is the most criticalvariable (34, 58). The area is a complex one; the fact thatconflicting results were obtained by Phillips and Kaye (113-115, 151, 152) and Gilbert et al. (75), on the one hand, andKereluk et al. (117) and Ernst (58, 59), on the other, is areflection of the diverse test procedures used. The first grouprecommended the use of a relative humidity (RH) of between30 and 40%, whereas the latter presented data to show thatsporicidal efficacy increased with increasing RH. The low-level RH recommendations were based on work in whichspores and their carrier materials were allowed to equilibratewith the RH of the test environment. The second group ofworkers were more interested in practical industrial applica-tions, and the spore test pieces were below equilibrium forthe moisture content of the load against the RH of thesterilizing environment.

In a model theory (59) put forward to explain theseconflicting data, spores are characterized with respect totheir immediate environment and relative moisture contentas compared with the gross environment surrounding them.The basis of this theory is that water molecules carry EtO toreactive sites; thus, in an environment with a relatively lowmoisture content with respect to the reactive site, thedynamic exchange must be directed outward, i.e., from thespore. The movement of EtO gas is thereby impeded, andthe macromolecules of the cell are less amenable to alkyla-tion. When the moisture content of the immediate environ-ment increases, the equilibrium condition arises, which isintermediate in effectiveness. As the environmental watercontent rises further, the dynamic movement of water isdirected towards the active site (the spore), the most idealsituation in practice. In the case of a relatively dry spore andlow-RH environment, there is little exchange of moistureinto and out of the spore, a situation very limiting forsterilization in practice. As the water content of the sporeand of the environment increases, a relatively wet spore andhigh RH are obtained, thus resulting in a zone of highmoisture which would have a diluting effect on EtO gas,reducing its availability to the spore (58, 59). This would bethe situation in those experiments (75, 113-115, 151, 152) inwhich the RH is above 40%, with an intermediate RH thusrepresenting the optimal RH of ca. 32%, designated by theseworkers.

(iv) Activity depends on the type of organism. Contrary tothe situation with most liquid biocides, to which spores areoften several thousandfold more resistant than nonsporulat-ing bacteria, spores are generally only some 2- to 10-foldmore resistant to EtO gas than are vegetative organisms (35,151, 152, 173). Spores of the thermophile B. stearothermo-philus and of certain other organisms may, in fact, be lessresistant to EtO than some vegetative bacteria such as S.aureus, Enterococcus faecalis, and Deinococcus radiodu-rans.

IP-Propiolactone. ,B-Propiolactone is not widely used as asporicidal agent (39). It exists as a colorless liquid at roomtemperature, boils at 1630C, and may be vaporized in aspecial atomizer. P-Propiolactone is noninflammable, haslow penetrating powers, and is claimed to be carcinogenic.

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TABLE 6. Neutralization of sporicidal and sporostatic activity

Sporicidal agent Neutralizing agent Comment

Glutaraldehyde Glycine ) Better then dilution or sodium bisulfite (could be toxic togerminating and outgrowing cells)

Formaldehyde Glycine

Hypochlorites Sodium thiosulfate Thiosulfate toxic to some streptococci; unlikely to be toxicto 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 functionof the concentration and the time and temperature at whichit is used (34, 35, 39, 99, 171-173). The temperature coeffi-cient, Q10, in the range of -10 to +25TC is 2 to 3 (99). As withEtO, however, the single most important factor determiningits sporicidal potency is water vapor, and for optimumactivity the RH should be kept above 70 to 75%. Again,however, it is not necessarily the atmospheric RH that isimportant, but the moisture content and location of water inthe bacterial cell. B. subtilis var. globigii spores equilibratedto 98% RH are readily killed by P-propiolactone at an RH of45%, an RH at which the spores are not usually susceptible.However, only a 2-log reduction (ca. 99% kill) is achievedwhen spores equilibrated to 75% RH are exposed to thisagent at 45% RH, and a small percentage of spores precon-ditioned at 1% RH is thereafter very resistant to ,-propio-lactone at 75% RH (99).

Other gases. Other gases with sporicidal activity includepropylene oxide (reviewed in reference 173) and ozone. Theformer is bactericidal and sporicidal, but less so than EtO,and is allowed to be used in the food industry. As with EtO,its activity depends on concentration, on time and tempera-t'ure, and especially on RH.Ozone has bactericidal and sporicidal properties, but its

instability and other undesirable properties were considered(99) to render it unsuitable for use as a gaseous disinfectant.However, more recent studies (63, 69) have demonstratedthe sporicidal activity of the gas, especially under acidic pHconditions against spores (B. cereus, C. perfringens, and C.botulinum) of importance in food processing.

RECOVERY AND REVIVAL OF INJURED SPORES

When exposed to chemical or physical agents, microor-ganisms may be inhibited, sublethally injured, or irreversiblydamaged, i.e., killed (7). In the laboratory, several types oftests are available for examining sporostatic and sporicidalactivity. In essence, the sporostatic tests involve determin-ing the MIC of a chemical agent, i.e., the lowest concentra-tion preventing germination or outgrowth or both. Practicaldetails can be obtained by consulting a forthcoming paper(A. D. Russell, B. N. Dancer, and E. G. M. Power, Soc.

Appl. Bacteriol. Tech. Ser., in press). It must be noted thatglutaraldehyde interacts strongly with nutrient media (80,182) and that this may present an erroneous impression as toits apparently low sporostatic (or bacteriostatic) activity.

Sporicidal evaluations may be of several types (8, 11, 36,71, 77, 85, 161, 173, 190, 202, 203, 227; Russell et al., inpress). Sporicidal activity can be tested against spores inliquid medium or suspended on appropriate carriers. What-ever method is adopted, and irrespective of whether quan-titative (survival counts) or qualitative (extinction) assess-ments are made, an appropriate method must be used todetermine spore survival (173). It cannot be emphasized toostrongly that adequate neutralization (quenching) of the testchemical must be achieved to prevent sporostasis occurringin subculture media and, consequently, false-negatives (37,78, 161, 173, 176). In brief, neutralization involves (i) dilutingthe biocide in the recovery medium to a level at which itceases to have inhibitory activity, (ii) incorporating into therecovery medium a neutralizing agent (antidote) that specif-ically inactivates the biocide and is itself nontoxic to germi-nation and outgrowth (177), or (iii) removing the biocide bymeans of a membrane filtration technique, followed bywashing the membrane in situ (with, if necessary, an appro-priate neutralizing agent, e.g., with QACs [38, 181]), andthen placing it on the surface of a solid nutrient medium.Suitable neutralization procedures for specific sporicidalagents are summarized in Table 6. It must be added thatsodium thioglycolate, widely used as an ingredient of anaer-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-

ature, normal (control, untreated) spores usually germinatevery rapidly, a process complete within 30 to 60 min. Sporesthat have been damaged, however, invariably require longperiods to repair this injury (7, 49, 50, 90, 159, 173, 200, 201,228), and for this reason it is prudent to prolong the incuba-tion period well beyond the usual period of, say, 48 h at(usually) 37°C; suboptimal incubation temperatures shouldalso be examined, as should the effect of composition andpH of the recovery medium (134).The revival of chemically injured spores may also be

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TABLE 7. Revival of bacterial spores exposedto sporicidal agents

.poricie.Posttreatment revival Refer-Sporicide procedures ence(s)

Formaldehyde Heat activation (60-90'C), plating 201

Glutaraldehyde UDS ± sonication, incubation in 76GML, plating in TSA

Heat (50-90'C), dilution in GM or 76GML, plating in TSA

NaOH or KOH, plating 49, 154

Povidone-iodine Incubation in GML, plating in TSA 76

a UDS, Urea plus DTT plus sodium lauryl sulfate; GM, germinationmedium; GML, germination medium plus lysozyme; TSA, tryptose soy agar.

achieved by specific procedures. It has been claimed thatsubjecting formaldehyde-treated spores of B. subtilis to aposttreatment heat shock at temperatures of 60 to 90'Cenables most of the supposedly killed spores to revive (200,201). A very small proportion of glutaraldehyde-exposedspores of various Bacillus spp. can be revived when, follow-ing neutralization of glutaraldehyde with glycine, the sporesare treated with alkali (49, 153). The rate of NaOH-inducedrevival is ca. 10-6 (i.e., CFU per milliliter of glutaraldehyde-exposed, NaOH-treated spores/unexposed, NaOH-treatedspores), obviously a low value but one of potential impor-tance (49). Experiments designed to distinguish betweengermination and outgrowth in the revival process haveestablished that sodium hydroxide (range, 10 to 50 mM;optimum, 20 mM) added to glutaraldehyde-treated sporesincreased the potential for germination. In contrast, B.subtilis spores which are allowed to germinate before expo-sure to low concentrations of glutaraldehyde and then tosodium hydroxide are inhibited at the outgrowth phase to amuch greater extent than germinated spores treated with thedialdehyde without subsequent alkali exposure (153). So-dium hydroxide can be replaced with potassium hydroxideor, to a lesser extent, sodium bicarbonate. The use of 2%(wt/vol) glycine (37) (Table 7) as an inactivator of glutaral-dehyde is of paramount importance in these revival studies.Alkali-induced revival of spores exposed to another dialde-hyde, glyoxal, has also been found with slight revival afterexposure to Gigasept (containing succinaldehyde plus form-aldehyde) but not to formaldehyde alone (E. G. M. Power,B. N. Dancer, and A. D. Russell, Lett. Apple. Microbiol., inpress). This interesting phenomenon could be related to thefar more damaging effects on the spores of glutaraldehydethan formaldehyde, with a consequent greater potential forrevival of the former than the latter.Some revival of glutaraldehyde-treated spores can be

achieved by means of a posttreatment heating (76, 154), butthe extent of this revival (maximum, two- to threefoldincrease in viable count achieved at 57°C) is less than byalkali treatment and also markedly less than that reportedwith formaldehyde (201). Coat-removing agents fail toachieve any revival (154) despite reports that glutaralde-hyde-treated spores damaged by one such treatment arecapable of germination (76). Lysozyme, either used beforeplating or incorporated into recovery media, is likewiseineffective, and a combination ofNaOH and lysozyme has aslight, but noticeable, deleterious effect on colony counts(154). To determine whether alkali induces protein release,

B. subtilis NCTC 8236 spores have been treated with glutar-aldehyde (which was then neutralized with glycine), washedwith buffer, and exposed to 20 mM NaOH for 10 min; therelease of protein was then determined chemically: only ca.1 pFg was released by alkali treatment (49). It is, of course,possible that a small number of spores may possess a higherthan average resistance to glutaraldehyde and become su-perdormant (86, 90) rather than damaged, so that they areable to germinate only under extreme conditions. Lysozymemay facilitate germination of damaged spores (90), althoughthis phenomenon applies mainly to thermally injured spores(see later results with hypochlorites, however). Gorman etal. (76) reported that increased survivor counts of iodophor-treated B. subtilis spores were obtained following exposureto lysozyme.

Information on the revival of chemically damaged sporesis still, on the whole, lacking. From an academic or theoret-ical point of view, considerable data may be generated aboutmechanisms of sporicidal action (64, 65), and thus studies onrevival are always worthwhile. Likewise, any assessment ofsterility must take into account conditions for repair ofinjured, but still viable and potentially harmful, bacterialspores. In the practical context, the studies from this labo-ratory with glutaraldehyde-damaged spores described abovehave used severe revival conditions that are unlikely to beencountered in practice; furthermore, an injured spore undersuch circumstances is effectively dead if it cannot germinateor outgrow. Nevertheless, there might be some small risk inoverestimating the sporicidal efficacy of glutaraldehyde.Spores have been recovered after 24-h exposure to thedialdehyde, whereas the Association of Official AnalyticalChemists test (8) recommends a 10-h exposure and muchshorter exposure periods are commonly encountered, par-ticularly in the hospital environment where time is often at apremium. An additional point is that in our studies (76, 153)only freshly activated glutaraldehyde solutions were used,whereas in practice older solutions are frequently used, withsome deterioration occurring (155). Nevertheless, mattersshould be kept in perspective, and Babb et al. (11) havestated that a 3-h treatment with 2% alkaline glutaraldehydeshould be sufficient for practical purposes to achieve asporicidal effect, especially as bacterial spores are onlyinfrequently found on clean medical equipment.The situation with formaldehyde is potentially more

alarming. Although alkali treatment does not revive formal-dehyde-treated spores (Power et al., in press), the studies ofSpicher and Peters (200, 201) suggest that the sporicidalactivity of this monoaldehyde might well have been overes-timated. Confirmation or rebuttal of their findings is awaitedwith interest.

Injured bacterial spores might be of concern in foodmicrobiology. Hypochlorites are used as sanitizers and altergermination responses of C. botulinum spores (65). In addi-tion, C. bifermentans spores are sensitized to lysozyme-induced germination following treatment with hypochlorite(237). Exposure of spores to EtO or H202 may alter require-ments for growth (64). Cook and Pierson (41) have pointedout that conditions used to enumerate spores in foods mightnot be optimum for germination and outgrowth of all sporesand that injured spores must be considered in this context.

SPOROGENESIS, SUSCEPTIBILITY, AND RESISTANCE

Sporulation, germination, and outgrowth are complexprocesses in the overall life cycle of Bacillus and Clostridiumspp. Differing responses to biocides are shown at different

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SPORICIDAL AGENTS 107

stages, and these aspects will be considered, as some usefulinformation can be obtained about the mechanisms of sporeresistance.

Sporulation

Sporulation is a multiphase process leading to the devel-opment of a spore from a vegetative cell. The stages in-volved (57, 118, 119) can be summarized as follows. Stage 0is the vegetative cell, stage I the presporulation phase (inwhich DNA is present as an axial filament), and stage II isthe septation phase in which asymmetric cell formationoccurs. Engulfment (encystment) of the forespore takesplace in stage III and cortex formation between the inner andouter forespore membranes commences in stage IV, withsynthesis of spore coats, dipicolinic acid, and uptake of Ca2+in stage V. Spore maturation occurs in stage VI, with thecoat material becoming more dense and refractility increas-ing. Lysis of the mother cell and liberation of the maturespore take place in stage VII. Clearly, there are severalstages at which antibacterial agents could act or, conversely,when resistance to such agents could arise. Sporulation(Spo-) mutants which are unable to develop beyond agenetically determined point (98, 108, 109) are of consider-able value in correlating structural changes, biochemicalcharacteristics, and susceptibility or resistance to specificbiocides. A practical consideration of these aspects is beingpublished elsewhere (Russell et al., in press).

Disinfectant-induced structural changes in fully developedspores have been described (123, 168, 189), but these havenot been fully related to their biochemical effects on sporu-lating cells. Thus, the mechanism of action (see later section)of many sporicidal agents is still often poorly described. Incontrast, the mechanisms of spore resistance to biocides arebetter understood, and these aspects will be consideredhere.

Resistance of bacterial spores (209) can be examined by (i)comparing the response of wild-type and Spo- mutants; (ii)using other mutants, e.g., conditional cortexless mutants ofB. sphaericus (103, 104); or (iii) comparing "normal" andcoatless forms of a spore.Development of resistance to biocides and antibiotics

during sporulation (82, 83, 130, 210) has been known forsome time. Experiments designed specifically to associatechanges in cell structure with altered responses to biocidescan yield useful information in this area. There is a need,however, to correlate these structural changes more accu-rately with biochemical changes in the spore. Useful mark-ers for monitoring the development of resistance are toluene(resistance to which is an early event), heat (intermediateevent), and lysozyme (late event) (108-111). In studies witha wild-type B. subtilis, strain 168, and its Spo- mutants, wehave demonstrated (156a, 188) that resistance to chlor-hexidine occurs later than that to toluene and at about thesame time as heat resistance, whereas glutaraldehyde resis-tance is a very late event, occurring after the development oflysozyme resistance (Table 8). Some 12 or so polypeptidesare found in the spore coat of B. subtilis (108-111); these aresynthesized at different times and are incorporated into thespore at stages V and VI. It has been suggested (108) thatone polypeptide of molecular weight 36,000, which is formedvery late in sporulation, may have a direct role in conferringresistance upon the spores. The development of glutaralde-hyde resistance, however, is unlikely to result from thedeposition of specific spore coat proteins because of thehighly reactive nature of the dialdehyde molecule (Power

TABLE 8. Onset of resistance to antibacterial agentsduring sporulationa

Sporulation SporulationAgent stage at which stage at which Commentresistance resistance is

appears fully developed

Toluene Late stage III Early stage IV Early eventChlorhexidine Stage IV Stage V IntermediateHeat Stage V Stage VI IntermediateLysozyme Middle of Stage VI Late

stage VGlutaraldehyde Late stage V Stage VI corm- Very late event

pleteda Based on Power and Russell (in press) and Shaker et al. (188).

and Russell, in press). Nevertheless, in general terms, theidea of attempting to correlate resistance with a specificcomponents) of the spore coat is an attractive one andshould be subjected to further experimentation.The increased resistance occurring during sporulation may

thus be related to the stage of spore development (Table 8).In many instances, the chemicals studied, e.g., xylene,toluene, or benzene, have been organic solvents rather thanpreservatives or disinfectants. Resistance to chloroform andto phenol, however, develops late in the sporulation process(12, 130), and that to methanol and ethanol occurs at thesame time as resistance to other alcohols, such as octanoland butanol. Alcohol-resistant sporulation mutants of B.subtilis can sporulate in the presence of alcohols at afrequency of 30 to 40% (24).

In conditional spore cortexless mutants of B. sphaericusdeficient in the synthesis of meso-diaminopimelic acid (Dap),the muramic lactam (and hence cortex) content increaseswith an increase in exogenous meso-diaminopimelic acid(103, 104). Characteristic spore properties have been foundto be associated with different amounts of cortex; e.g., ca.25% of maximum cortex content is necessary for the spore topresent resistance to octanol but ca. 90% is necessary toshow heat resistance. Such Dap- mutants might thus beuseful in studying mechanisms of spore resistance to bio-cides, although as pointed out by Waites (224), changesother than variations in cortex development might occurelsewhere in the spore which must be considered beforeascribing resistance solely to the cortex.Probably the most detailed approach to studying resis-

tance of spores has involved the use of spore coatless forms(46, 62, 63, 69, 82, 88-91, 93, 124, 126, 132, 189, 194, 212,224-228, 231, 237). Methods of removing one or both sporecoats have been described in detail by Nishihara et al.(141-143). Coats may be extracted by using 2-mercaptoeth-anol, sodium lauryl (dodecyl) sulfate, dithiothreitol (DTT),and urea. Treatments consist of urea plus DTT plus sodiumlauryl sulfate, urea plus DTT, urea plus 2-mercaptoethanol,and sodium lauryl sulfate plus DTT. Of these treatments,urea plus DTT plus sodium lauryl sulfate is usually consid-ered the most satisfactory. Both lysozyme and nitrous acidor sodium nitrite are effective against coatless, but notnormal, spores, although pretreatment of the coatless sporeswith glutaraldehyde reduces the extent of this activity con-siderably (82). The role of the spore coat in resistance ofspores to various antibacterial agents is summarized in Table9. Hydrogen peroxide itself will remove coat protein from C.bifermentans (231); however, removal of coat protein byDTT before spore exposure to peroxide markedly increasesits lethal effect, whereas B. cereus spores are much less

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TABLE 9. Mechanisms or site of resistance of bacterialspores to chemical agents

Antibacterial Spore Commentagent component

Alkali Cortex

Lysozyme Coat(s) 1Hypochlorites Coat(s) UDS spores highly sensitiveGlutaraldehyde Coat(s)Iodine Coat(s)

Hydrogen peroxide Coat(s) Varies with strain

Chlorhexidine Coat(s) UDS spores more sensitivethan "normal" spores

Ethylene oxide Coat(s) Exact relationship unclear

Octanol Cortex | Dap- mutants of B. sphaeri-Xylene Cortex J cus more sensitive

a Dap, meso-Diaminopimelic acid.

affected (226). The spore coat is thus likely to confer a

protective effect against peroxide to the former but not to thelatter spores, thereby demonstrating that varying responsesoccur with different sporeformers and that the response maybe associated with different composition and structure of thespore coat(s). Spores treated with urea plus DTT plussodium lauryl sulfate are highly susceptible to glutaralde-hyde, iodine, hydrogen peroxide, ozone, and chlorine (63,76, 82, 174). Thus, even with agents that are known to beactively sporicidal, the coats play a role in limiting intracel-lular penetration.

Nevertheless, the role of the spore coats in resistance toEtO is unclear. In B. subtilis, removal of the coats increasesspore sensitivity (129). However, resistance to EtO of sporesof B. cereus strain T pretreated with alkaline DTT remainsunchanged (46). Furthermore, B. subtilis 4673 (a mutant ofstrain 4670) with defective coats and outer coat layersthinner and more diffuse than 4670 is more resistant to EtOthan is 4670. Conversely, strain EV15 which overproducescoat material, thereby possessing an abnormally thick mul-tilayered coat, has an exceptionally high resistance to EtO(46). On the other hand, these findings imply that theexpected increased permeability to EtO in strain 4673 doesnot occur and, on the other, that increased resistance to EtOis associated with excessive coat production. EtO is a

comparatively small molecule, but molecular size appears tobe of little consequence where coat impermeability is con-cerned. For example, H202 (molecular weight, 34), ozone

(03; molecular weight, 48), and chlorine dioxide (C102;molecular weight, 67.5) are all small molecules, yet spore

coats are considered a primary protective barrier to theirentry (63, 69, 70, 123).The spore coat appears to act as a permeability barrier to

chlorine (123, 224, 226, 237), since coatless spores are

rendered more permeable to hypochlorites. Chlorine willitself remove coat protein and allows lysozyme to initiategermination (237). Sodium hydroxide increases the perme-

ability of bacterial spores to germinants, and the potentiationof hypochlorite action by sodium hydroxide (42) may be theresult of the effect of the alkali on spore coats from whichprotein is removed (93), although the cortex is alkali resis-tant (123).

Germination

Activation is a treatment resulting in a spore which ispoised for germination but which still retains most sporeproperties; activation is thus responsible for the breaking ofdormancy in spores, but is reversible. In contrast, germina-tion itself is an irreversible process and is defined as a changeof an activated spore from a dormant to a metabolicallyactive state within a short period of time.The first biochemical step in germination is the biological

trigger reaction. This initiation process can be induced bymetabolic or nonmetabolic means, although it is now gener-ally believed that the trigger reaction is allosteric in naturerather than metabolic, because the inducer does not need tobe metabolized to induce germination. Initiation of germina-tion is followed rapidly by various degradative changes inthe cell, leading within a short period of time to outgrowth.These changes include (87, 217) (i) a decrease in heatresistance accompanied by changes in staining properties,(ii) a decrease in refractility whereby phase-bright spores(Fig. 2a and b) become phase dark (Fig. 2b and c), (iii) adecrease in dry weight, and (iv) a decrease in optical density,a comparatively late event in germination (205), although it isa widely used method for measuring germination. Inhibitionand control of spore germination are important consider-ations in many fields, including food preservation (74, 193),although dormancy may be a problem (86).

Several antibacterial agents are known to inhibit germina-tion. These include alcohols, aldehydes, phenols andcresols, parabens, sorbic acid, and mercuric chloride (2, 3,47, 66, 68, 100, 128, 148, 149, 156, 166, 168, 171, 172, 181,190, 193, 195, 198, 219, 220, 223, 234; B. M. Lund, Ph.D.dissertation, University of London, London, England,1962). This inhibition (Table 10) occurs at concentrationsthat are closely related to those that inhibit the growth ofvegetative bacteria.The effects of inhibitors of spore germination may be

reversible. This is apparent from the results of studies withphenols (128, 148, 149, 181), formaldehyde (219), alcohols(220), and parabens (157, 234). These findings suggest a fairlyloose binding of these agents to a site(s) on the spore surfacesince mere washing is often sufficient to dislodge the inhib-itor.Mercuric chloride is a powerful inhibitor of the germina-

tion of spores of C. botulinum type A (2) and ofBacillus spp.(92, 100, 223). It appears to inhibit some reactions ingermination before the loss of heat resistance but not thesubsequent release of peptidoglycan (223). In contrast, anorganomercurial compound, phenylmercuric nitrate, hasbeen shown (148, 181) to have little effect on the germinationof B. subtilis spores but a pronounced inhibitory effect onoutgrowth.Germination (Ger) mutants of B. subtilis 168 deficient in

the initiation of germination 135, 184), could be of value instudying the mechanism of action of antibacterial agents butdo not, as yet, appear to have been studied in this context.

Glutaraldehyde exerts an effect early in the germinationprocess (153, 156). This belief is based on several experi-mental approaches, a very recent one (156a) involving theeffect of the dialdehyde on the uptake of L-[14C]alanine to B.subtilis spores. This germinant is considered to act bybinding to a specific receptor to the spore coat (184), andonce spores are triggered to germinate, they are committedirreversibly to losing their dormant properties (205). Thealdehyde could inhibit germination by (i) reducing L-alanineuptake as a consequence of competition for binding sites on

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(a) (b) (c)

FIG. 2. Changes during spore germination examined by phase-contrast microscopy. (a) Mature, phase-bright spores; (b) development ofphase-dark forms; (c) germination complete with full conversion to phase-dark cells.

the spore, (ii) preventing passive diffusion of L-alanine intothe spore, (iii) sealing the spore surface, or (iv) inhibiting theL-alanine-induced trigger reaction of germination by a later,as yet unexplained, mechanism (Fig. 3a to d, respectively;Table 11). In our experiments, despite earlier claims to thecontrary (112, 238-242), D-glucose has no significant effecton L-alanine uptake. These other workers, however, calcu-lated the binding affinity of glucose solely on the loss of heatresistance and turbidity of germinated spores rather than bydirect methods. Glutaraldehyde-treated spores retain theirrefractility, having the same appearance under the phase-contrast microscope as normal untreated spores (Fig. 2a),even after subsequent incubation in germination medium.The observation suggests that inhibition occurs very early

in the germination process. At concentrations up to 0.1%(wt/vol), both acid and alkaline glutaraldehyde inhibit ger-mination, but not L-[14C]alanine uptake, and therefore pre-

TABLE 10. Inhibitors of germination and outgrowth

Process Inhibitor Comment

Germination Glutaraldehyde Probably inhibitstrigger mechanism

Sorbic acid Inhibitor of triggermechanism?

Formaldehyde, alcohols Diverse group,phenols, parabens probably differentmercuric chloride J sites of actionSodium thioglycolate Caution needed with

recovery media

Outgrowth QACs, chlorexhidine No or little effect onEtO, organomercurials germinationHypochlorites IGlutaraldehyde Even more effective

at these stagesSorbic acid Multiple sites of

inhibition

vent the trigger reaction by some unexplained means. Athigher glutaraldehyde concentrations (01. to 1%, wt/vol),uptake of L-alanine is significantly reduced, presumably theresult of a sealing effect by the aldehyde on the sporesurface. Spores do not concentrate L-alanine and uptakeproceeds rapidly without the necessity for an energy-depen-dent active transport system, demonstrating that the dor-mant spore is freely permeable to the amino acid, whichenters by simple diffusion (53, 154).The effects of inhibitors of germination have been widely

reported, but it is often not known at what stage of germi-nation an inhibitor is active (193). Because of the nature ofthe germination process, the only types of antibacterialagents that are effective are those that inhibit the triggerreaction and those that prevent the degradative processes.Many antibacterial agents are known to affect the opticaldensity changes that occur in germination. A decrease in

SPORE

L-AI

Trigger forgerm i nation

FIG. 3. Possible sites of action of an inhibitor of the triggermechanism in spore germination.

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TABLE 11. Effect of glutaraldehyde on the germinationtrigger mechanism

Process Effect of glutaraldehyde

Decrease in optical Low concn (<0.1%) inhibitory, con-density siderably below sporicidal levels

(2%)

L-['4C]alanine binding to Inhibition, but only at high aldehydespores concn

Phase darkening of Low concn (<0.1%) prevents (seespores Fig. 2)

Outgrowth of previously Even more inhibitory than vs germi-germinated spores nation

optical density is a late event in germination and is notsuitable for studying the initial reactions (193). Thus,whereas many compounds are likely to prevent the degra-dative processes, it is unclear whether the trigger reaction isalso affected. Even a procedure involving a short period ofexposure to the inducer, e.g., L-alanine, followed by moni-toring of the fall in optical density is considered to beinappropriate, as is one involving the release of 45Ca (205).Suitable methods are the direct one, described above, in-volving the uptake of labeled L-alanine, and one in whichspores are exposed to L-alanine for a very short period oftime. The reaction is then stopped by adding an excess of itscompetitive inhibitor, D-alanine. The commitment to germi-nation is then measured by counting the conversion ofphase-bright spores (Fig. 2a) to phase-dark spores (Fig. 2c).This technique has been used to study the effect of sorbate,an effective inhibitor of germination (198). Busta and hiscolleagues (21, 198) have concluded that sorbic acid does notcompete with L-alanine for a common binding site on thebacterial spore, so that inhibition occurs after germinantbinding (41).

Alcohols inhibit the L-alanine-initiated germination of B.subtilis spores, suggesting that this inhibition results from aninteraction of a hydrophobic region in or near the L-alaninereceptor site on the spore with the hydrophobic group on thealcohol (242). Such interaction is presumably of a weaknature, because (as pointed out earlier) the inhibition ofgermination by alcohols is reversible. Unfortunately, only anoptical density technique was used in these studies. The heatactivation of C. perfringens spores at a temperature range of70 to 80'C in water is enhanced in the presence of alcohols(43, 44). The concentration of a monohydric alcohol toproduce optimum spore activation is inversely related to itshydrophobic character.Other inhibitors of germination include sodium bicarbon-

ate (15, 45) and cyclic polypeptide antibiotics (96). Antibiot-ics are outside the scope of this paper, but the experimentalapproach involving morphological changes and inhibition ofmacromolecular syntheses has yet to be applied to many

biocides. B. brevis Nagano wild type produces the antibioticgramicidin S, which inhibits germinating spores (138). Aparticularly interesting property of this organism is thatgermination-initiated spores retain their resistance proper-

ties (48), and it is likely that this property could be studiedfurther with a range of biocides.

OutgrowthOutgrowth is defined as the development of a vegetative

cell from a germinated spore and takes place in an orderly

manner when germination is carried out in a medium thatsupports vegetative cell growth. After germination, germi-nated spores become swollen and shed their coats to allowthe young vegetative cells to emerge, elongate, and divide.Of the macromolecular biosynthetic processes occurringafter germination, RNA synthesis is the first, followedclosely in Bacillus spp. by the onset of protein synthesis,with DNA synthesis occurring some time later. Duringoutgrowth, all types of RNA are synthesized. Cell wallsynthesis commences after RNA and protein but beforeDNA and coincides with swelling of the germinated spore.

Several antibacterial agents act at the outgrowth ratherthan the germination stage (Table 10). These include QACs,organomercurials, chlorhexidine, and EtO (35, 148, 181, 190;Lund, Ph.D. dissertation), the first three of which aresporostatic agents, with EtO a sporicidal compound. QACsbind strongly to spores, and simple washing procedures willnot remove them (38). QAC-treated spores which are mem-brane filtered are still prevented from undergoing outgrowthwhen transferred to an appropriate growth medium (38, 181),and a neutralizing medium must be used in conjunction withmembrane filtration.The parabens and similar substances inhibit germination at

sporostatic concentrations. Outgrowth is prevented at higherconcentrations. High concentrations of hypochlorites arenecessary to prevent spore germination, whereas moderateconcentrations markedly retard outgrowth and low concen-trations have only slight effects on either (237). Sublethalconcentrations of EtO inhibit outgrowth but not germination(159), and resistance of spores to EtO does not decreaseduring germination (46, 47). Hydration of the spore core andalteration of spore coat layers do not therefore appear to belinked to an increased susceptibility. Even spores exposed tohigh EtO concentrations can germinate freely under a vari-ety of conditions but will not outgrow (47), but asparagineacts as a germinant for untreated but not EtO-treated spores.A former food preservative, sodium nitrite, has been the

subject of heated debate as to how it exerts its antimicrobialactivity (173). It does not affect spore germination and in factinduces germination, but only at high concentrations (2, 3,125). Nitrite inhibits postheating germination or outgrowthor both, heat-injured spores being rendered more susceptibleto the salt (105).

It is apparent from this section and the preceding one thatmost sporostatic compounds inhibit either germination oroutgrowth. (An exception to this general statement is glu-taraldehyde, low concentrations of which inhibit both pro-cesses [154]). What is not clear is why this should be so.There has been little basic research to explain why onecompound should, for example, inhibit the degradative pro-cesses associated with germination, whereas another com-pound has no effect at this site but inhibits the later stage ofoutgrowth. Figure 4 summarizes the effects of antibacterialagents on germination and outgrowth, as well as detailingstages during sporulation at which specific resistances de-velop.

OVERCOMING SPORE RESISTANCEBacterial spores can pose a problem insofar as the activity

of chemical agents is concerned. It is obviously essential touse appropriate concentrations at the optimum pH for asufficient period of time to ensure a sporicidal effect. Thereare, however, means available for achieving the same, or anenhanced, response. These involve a combination of achemical and a physical process or of two chemical agents(1).

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SPORICIDAL AGENTS 111

Glut, -SPORE----... .IGlut?Lys . t Trigger.*-- SA?

CHA- do Degradative.. Other germinationTol- processes inhibitors?

CELL CULTURE GER INATION

Several. , V GlutOinhibitors? AsGlt 0

OUTGROWTHFIG. 4. Summary of possible effects of some antibacterial agents

on germination and outgrowth and of the development of resistanceduring sporulation. Glut, Glutaraldehyde; SA, sorbic acid; Tol,toluene; CHA, chlorhexidine diacetate; Lys, lysozyme; EtO, eth-ylene oxide.

The bactericidal and sporicidal activity of a biocide in-creases with increasing temperature. For example, the tem-perature coefficient (0) per 1C rise in temperature for thephenolic agent chlorocresol is 1.1 (33). If the temperature isincreased from 20 to 1000C, then assuming that 0 is the sameover the entire temperature range, the activity increases by>2,000-fold (0100-20 = 1.180 = 2,104). Use of this principlewas, until 1988, made in the United Kingdom, where "heat-ing with a bactericide" was one official (pharmacopoeial)method of sterilizing certain parenteral and ophthalmic prod-ucts. Differences between acid and alkaline glutaraldehydeforms disappear at temperatures above about 400C (28-30,213). A process that has been promising results is the use ofsaturated steam at subatmospheric pressure in the presenceof formaldehyde (173). The complicated effect of heat andsodium nitrite has been described (105) and reviewed (173).Acid heat treatment is an important means of controllingspores in food, spores being more susceptible to heat at lowpH (20). Spores can, in fact, exhibit a base exchangebehavior which will reduce, restore, or enhance their ther-mal resistance. Sensitization involves converting them to thehydrogen (H) form, which can be transformed into theresistant calcium (Ca) form by treatment with calcium ace-tate at pH 11. H-form spores are also more susceptible topropylene oxide but less so than the Ca form is to glutaral-dehyde (210, 214).Another physicochemical process is the use of glutaralde-

hyde with ultrasonics (28-31, 192). Although ultrasonicwaves themselves possess little sporicidal activity (andhave, in fact, been used to separate spores and vegetativecells), they have been claimed to potentiate the sporicidalactivity of acid glutaraldehyde at 60'C and of hydrogenperoxide but not of iodophors. The QAC benzalkoniumchloride still had a poor sporicidal effect (31). A synergisticeffect of hydrogen peroxide used in conjunction with UVradiation has been shown to occur (17, 230).Combinations of chemical agents have also been studied.

Examples have already been provided of hypochlorites andmethanol (40, 51) and of glutaraldehyde with nonionic (28-31) or anionic (81) surfactants or with inorganic cation-anionic surfactant combinations (81). In many instances,however, the underlying reasons for this potentiation remainobscure. Glutaraldehyde has also been combined with phe-nol plus phenate, the combination being claimed (106, 107) tohave an enhanced sporicidal action. Alcohol is not spori-cidal, and only high concentrations (9%) of hydrochloric acidhave this property, whereas acid alcohol (1% HCl in 70%

TABLE 12. Mechanisms of action of some chemical agents

Antibacterial Site or mechanismagent of action

Alkali Inner spore coatChlorine compounds CortexEthylene oxide Alkylation of core protein and DNAGlutaraldehyde CortexHydrogen peroxide Spore core?Lysozyme Cortex (1, 1--4 links)Nitrous acid Cortex (at muramic acid residues)

alcohol) kills spores in 4 h (208). Even combinations of localanesthetics and preservatives are claimed to be sporicidal(1).

Clearly, no comprehensive studies have been undertakento determine what factors are involved in designing a processwith enhanced sporicidal activity. Also, reasons for anysynergism are often totally inadequate. This aspect is onethat could, with benefit, be addressed.

MECHANISMS OF SPORICIDAL ACTIONA considerable amount of information is available about

the ways in which bactericidal agents affect nonsporingbacteria (175). In contrast, mechanisms of sporicidal activityare poorly understood (236). The major reason for this isundoubtedly the complex nature of the bacterial spore, towhich may be added the possibility that an antibacterialcompound might have more than one actual or potential siteof action. While contributing to the overall lethal effect, thispossibility can complicate still further the unraveling of themechanism of sporicidal action.The spore does present several sites at which interaction

with an antibacterial agent is possible, e.g., the inner andouter spore coats, cortex, spore membranes, and core (Table12). Interaction with a particular site need not necessarilyimply, however, that this is associated with death of thespore or that there is only one site or target in the spore thatmust be inactivated. Practical considerations have recentlybeen described (Russell et al., in press).Data on uptake of an antibacterial compound to bacterial

cells are often considered a useful starting point in examiningthe mode of action of the compound. Studies on the uptakeof glutaraldehyde to different types of bacteria (156) haveshown that E. coli, B. subtilis vegetative cells, and S. aureusbind more aldehyde than do resting B. subtilis spores.Uptake increases during spore germination and outgrowthbut is less than to vegetative cells. The surface of bacterialspores is hydrophobic in nature (54). Low concentrations ofboth acid and alkaline glutaraldehyde increase this surfacehydrophobicity (156), presumably as a consequence of theextensive interaction of the dialdehyde with outer layers ofcells and spores (19, 84, 140, 160, 211). The greater spori-cidal activity of the alkaline form is not reflected by theuptake patterns but it is likely that acid glutaraldehyderesides at the cell surfaces, whereas the alkalinating agent,sodium bicarbonate, assists in the increased penetration ofthe alkaline form into the spore (120, 137). The major initialeffect of bicarbonate is believed to be on the outer layers ofspores of bacterial cell walls (79-81, 84, 137), although it willalso inhibit germination of Bacillus spores (15, 45). Glutaral-dehyde is thus likely to seal the outer layers of spores, anaction that would also be of importance in inhibiting sporegermination (154), with penetration at alkaline pH into thespore. Glutaraldehyde combines with amino acids (94, 182,

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235) and has been found to interact strongly with the cortexand spore protoplast, the latter prepared by Fitz-James'technique (61). Interaction with the cortex might be respon-sible for the sporicidal action of the aldehyde. Penetrationand reaction of glutaraldehyde with components of this layermay be assisted by the action of divalent cations (81, 84).

Other antibacterial agents also interact with the outerspore layers. Chlorhexidine diacetate increases spore hydro-phobicity (189) but is not sporicidal (188, 190) unless used athigh concentrations at high temperatures (77). As withQACs, therefore (38), it is likely that this cationic agentcombines strongly with spore coats, but is unable to pene-trate into the spore (169).

Hypochlorites solubilize the cell walls of nonsporing bac-teria and the spore integuments of B. megaterium spores(167). Separation of spore coats from cortex, followed bysequential dissolution of spore layers, has been described(123). Bacillus and Clostridium spores exposed to hypochlo-rites leak dipicolinic acid (56, 62, 123), suggesting an in-crease in spore permeability that can also be achieved byheat alone (18). The spore coat appears to act to some extentas a permeability barrier to chlorine (123, 224-226, 237),since the removal of protein from spore coats renders sporesmore susceptible to hypochlorites. These chlorine com-pounds will themselves remove coat protein, thereby allow-ing accessibility of cortex to lysozyme, which initiatesgermination (67, 237). Pretreatment of spores with sublethalconcentrations of chlorine renders the cells more susceptibleto mild heating (56). This effect may result from an alterationof spore cortex (237) since the cortex is believed to controlspore response to high temperatures (89-91). The cortexmay therefore be the major site of chlorine action, particu-larly since the removal of spore coats does not affect sporeviability.The mechanism of sporicidal action of hydrogen peroxide

has also been widely studied (4-6, 13, 14, 16, 17, 93, 121,166, 173, 174, 204, 215, 224-226, 229, 231). Hydrogenperoxide removes coat protein from C. bifermentans, butcoat protein removal (by DTT) from spores prior to peroxidetreatment markedly increases its effectiveness, although B.cereus is affected to a lesser extent. Sublethal levels ofperoxide increase the germination rate in C. bifermentans.Exposure of peroxide-treated spores to monovalent cationsor to increasing pH results in a complete loss of theirrefractility (231). H202 increases the lysis of spores of C.bifermentans in the presence of certain divalent cations suchas Cu2+, but the effect with other spores is less marked (16,17). Although C. bifermentans and B. subtilis var. nigerspores take up Cu2+ at about the same rate, only the sporeprotoplasts of the former bind these cations.

Activation of peroxide to hydroxyl radicals (-OH) is nec-essary for sporicidal action, which would explain the syner-gistic effect noted with the combined use of hydrogenperoxide and UV radiation (17, 228, 230). DTT-treatedspores of C. perfringens are much more susceptible toperoxide-induced lysis in the presence of Cu2+ ions than areuntreated spores (4), and, significantly, this lysis is reducedby -OH scavengers. Peroxide and Cu2+, alone, do notproduce lysis of cortical fragments but in combination in-duce lysis. It has been suggested (4) that peroxide may reactwith Cu2+ bound to cortex peptidoglycan, thereby generat-ing -OH radicals. These would be formed at the region nearthe germ cell wall and would be responsible for causingprotoplast lysis. DTT-treated C. perfringens spores undergogerminationlike changes followed by lysis when exposed toperoxide-generating systems (5).

Spores of C. bifermentans produced on different mediareact differently to hydrogen peroxide, the more resistanttypes having a thicker cortex and smaller protoplast (227,229). Peroxide has a marked effect on spore structure (123,231), the cortex becoming depleted or absent and the ribo-somes becoming disordered.Taken as a whole, the above findings suggest that hydro-

gen peroxide has an effect on the spore coats in someorganisms but that this in itself is insufficient to explain itssporicidal activity. Its major effects are undoubtedly on thecortex and core. The evidence to date implies tentativelythat the core is the major site of action, but further studiesare needed to substantiate this conclusion.Of the other sporicides, the mechanism of sporicidal

action of only one (EtO) has been examined in detail. Themode of action of iodine has, surprisingly, been little studied(173). It is considered to bind to bacterial protein (207, 208),but this vague attribute does little to explain how it killsspores. Formaldehyde is considered (208) to be sporicidalbecause it can penetrate into the interior of the bacterialspore. This monoaldehyde is an extremely reactive chemical(173, 179), combining with protein, RNA, and DNA, but thereasons for its sporicidal action remain somewhat obscure.EtO is an alkylating agent believed to inactivate bacterialspores by combining with various groups in proteins andnucleic acids (34, 35, 173, 174). B. subtilis spores exposed toEtO release considerably greater quantities of DNA, RNA,protein, and dipicolinic acid than do untreated spores (129),but EtO is not mutagenic to bacteria or spores (75, 99),unlike liquid sulfur mustard, an alkylating agent which is alsomutagenic.Exposure of spores to trichloroacetic acid alters their

viability and germinability and their response to heat andalkali but not to lysozyme (191). Trichloroacetic acid couldhelp provide useful data about mechanisms of action of otheragents.

MEDICAL AND OTHER USES OF CHEMICALSPORICIDES

Previous sections have dealt with the spectrum of activityand mechanisms of action and of bacterial resistance. Thissection will consider some uses of chemical sporicidal agents(see also references 73, 175, and 183).

Sporicidal Agents

Glutaraldehyde, one of the most potent sporicidal agents,is extensively used in the leather tanning industry and intissue fixation for electron microscopy and has numerousbiochemical applications (11, 84, 179). In the microbiologicalcontext, the dialdehyde has chiefly been used for the chem-ical sterilization of medical equipment which cannot besterilized by physical methods (84, 163, 185, 186, 216, 222).The main advantages claimed for its use as a chemosterilizerare (i) its broad spectrum of activity, especially good spori-cidal properties; (ii) its activity in the presence of organicmatter; (iii) its rapid antimicrobial action, although sporesare considerably less susceptible than nonsporing bacteria;(iv) its noncorrosive action towards metals, rubber, lenses,and most materials, although some formulations may notfulfil these criteria (9, 11); (v) its lack of harmful effects oncement or lenses of bronchoscopes, cystoscopes, or tele-scopes; and (vi) its ease of use. Nevertheless, its pungentand irritating odor to personnel over long periods of time isa distinct disadvantage (206). Rittenbury and Hench (164)

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and Haselhuhn et al. (95) recommended glutaraldehyde forthe cold sterilization of hemostats, cystoscopes, food con-tainers, and anesthesia equipment. The aldehyde has alsobeen found to be completely satisfactory for the routinesterilization of urological instruments and endoscopes (133,145) and has proved highly effective for the rapid and safedisinfection of gastrointestinal endoscopy equipment (216).Despite inadequate sterilization with glutaraldehyde, be-cause of the short time periods often used in hospitalpractice, infection transmission appears to be rare, presum-ably because very few potentially pathogenic spores are tobe found on cleaned endoscopes (9, 11). Bovallius and Anas(32) demonstrated the effectiveness of vapor-phase glutaral-dehyde for surface disinfection against sporing and nonspor-ing bacteria. In spite of its low volatility, it was moreeffective than formaldehyde.Formaldehyde is used as a solution and in vapor-phase

form. In the liquid phase, formaldehyde is used as a disin-fectant and as a general farm disinfectant (180). Low-temperature steam (without formaldehyde) for 10 min at730C is probably the most suitable method for disinfectingcystoscopes between patients (11). Longer periods of use oflow-temperature steam with formaldehyde are required forsterilizing laparoscopes and arthroscopes, but this damagesmost flexible fiber-optic endoscopes (11).

Chlorine-based products are used as food sanitizers, manyof which are designed to control bacterial spores (70).Hypochlorites are used as disinfectants in the dairy industry,for the disinfection of farm buildings (180), and as disinfec-tants in hospitals and food establishments (23). They havecertain advantages over glutaraldehyde (11) in that they killspores rapidly, so that instruments could be sterilized ratherthan disinfected between patients during busy endoscopysessions. They can, however, cause instrument damage. Theactivity of hypochlorites is reduced drastically in the pres-ence of organic matter, whereas organic chlorine compoundsare less susceptible (23). Chlorine dioxide does not formchlorinated organic compounds and is an effective sporicide,and its activity is not significantly affected by pH (70).

Concentrations required and conditions of use precludethe wide use of hydrogen peroxide as an effective sporicide.Nevertheless, peroxide has been used for sterilizing foodcontact surfaces, that is, in obtaining commercial sterility ofthe packaging material when rapid spore destruction isrequired. Its medical (e.g., for cleansing wounds and for eardrop formulations) and other uses do not usually rely on itssporicidal activity.EtO is mainly used as a chemical sterilizing agent (35,

173), but has also been used as a decontamination agent forarticles handled by tuberculosis patients. In the UnitedKingdom, it is one of the pharmacopoeial methods describedfor sterilizing powders, and Russell (173) has listed equip-ment that has been sterilized by EtO, in each instance withspores used as an indicator for satisfactory sterilization.These materials included various ophthalmic instruments,anesthetic equipment, heart-lung machines, disposable sy-ringes, and hospital blankets. A problem always associatedwith EtO is the possibility of toxic effects arising fromresidual EtO present in products (162). Although EtO gasdiffuses rapidly in open air, porous materials adsorb the gasduring the sterilization cycle in various amounts and thenrequire various poststerilization periods for desorption ofresidual gas.The antibacterial agents with sporicidal activity described

in this section, therefore, can be used as chemical sterilizingagents. Two points must be added, however: (i) they are

much less potent than thermal sterilization methods, espe-cially autoclaving, but often find a use in sterilizing thermo-labile equipment; (ii) it is somewhat arbitrary to consideronly their sporicidal activity, since many of the biocides arealso used in other environments, e.g., as disinfectants, or asdecontaminants in specific areas as with materials contam-inated with mycobacteria, human immunodeficiency virus,or hepatitis B virus.

Inhibitors of Germination and OutgrowthAntibacterial agents that are not sporicidal but instead

inhibit germination or outgrowth or both have uses that aredifferent from sporicides. Sporeforming bacteria are of par-ticular concern in food products (74, 127), especially whenthey are capable of surviving food-processing treatments, ofcausing food spoilage, and of being foodborne pathogens,e.g., C. perfringens, C. botulinum, and B. cereus (41, 69).Because of changes in the food itself (palatability andnutritional aspects), it is often impossible to destroy allspores that might be present. Consequently, specific antimi-crobial agents are often included to inhibit growth fromspores. Sodium nitrite delays, but does not prevent, botuli-nal outgrowth (41, 105), but its potential toxicity to humansis now well known (196, 197). Methyl and propyl parabensare commonly used in the food industry as preservatives,with the propyl ester more effective in inhibiting C. botuli-num growth and toxin production (171). Sorbic acid is aweak lipophilic acid widely used as a food preservative (127,168); it inhibits botulinal spore germination (there being aloss of heat resistance), a property that is pH dependent andappears at pH values of <6 (165, 196, 197). Potassiumsorbate delays C. botulinum growth and toxin production incured meats (64) and, when added to acidic foods, ishydrolyzed to sorbic acid. The acid is effective in theundissociated form (pKa, 4.75), and the maximum pH foractivity is ca. 6 to 6.5. In addition to the effect on germina-tion noted above, sorbate delays or inhibits the outgrowth ofC. botulinum spores (20, 21). Sorbate plus nitrite is aneffective combination also, but the need to eliminate thelatter as a preservative does not make this a viable proposi-tion.

Antibacterial agents are also used as preservatives inpharmaceutical and cosmetic products. Here, however, spe-cific sporeforming agents are not necessarily the majorproblem (172).

CONCLUSIONS

Comparatively few bactericidal agents are actively spori-cidal. The most important chemical sporicides are glutaral-dehyde, formaldehyde (liquid and vapor forms), chlorine-releasing agents, peroxygens, and ethylene oxide. Ozonemay become an important addition in the near future. Evenso, activity against spores is invariably considerably slowerthan against vegetative cells, and concentrations are higherfor a sporicidal action to be achieved. Bactericidal andbacteriostatic chemicals that are not sporicidal are usuallysporostatic, preventing spore germination or outgrowth orboth. Exact mechanisms of sporicidal activity and of sporeresistance have yet to be elucidated, and further studies areundoubtedly necessary.

In the clinical context, a rational approach to disinfectantsand sterilization, involving a consideration of both patientrisk and the treatment of equipment and environment (9, 10,60), is necessary. Moist heat is the preferred form of

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sterilization (usually at temperatures of 121'C or above) or,as low-temperature steam (e.g., 730C), of disinfection. Otherspecific methods of sterilization, such as ionizing radiation,dry heat, filtration, and gaseous chemical agents (ethyleneoxide and low-temperature steam with formaldehyde) areused when relevant. Chemical disinfectants (liquid chemicalsterilants) should only be used when other methods ofsterilization are inappropriate. Thus, glutaraldehyde is usedfor sterilization of medical equipment when heat cannot beused; for example, articles in a high-risk category may bethermolabile but require a process that is sporicidal.

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153. Power, E. G. M., B. N. Dancer, and A. D. Russell. 1988.Emergence of resistance to glutaraldehyde in spores of Bacil-lus subtilis 168. FEMS Microbiol. Lett. 50:223-226.

154. Power, E. G. M., B. N. Dancer, and A. D. Russell. 1989.Possible mechanisms for the revival of glutaraldehyde-treatedspores of Bacillus subtilis NCTC 8236. J. Appl. Bacteriol.67:91-98.

155. Power, E. G. M., and A. D. Russell. 1988. Assessment of 'ColdSterilog Glutaraldehyde Monitor.' J. Hosp. Infect. 11:376-380.

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156a.Power, E. G. M., and A. D. Russell. 1990. Uptake of L-14C-alanine to glutaraldehyde-treated and untreated spores ofBacillus subtilis. FEMS Microbiol. Lett. 66:271-276.

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