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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 arehighly 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)