Bacterial Spores and Chemical Sporicidal Agents A
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CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 99-119 Vol. 3, No. 2 0893-8512/90/020099-21$02.00/0 Copyright © 1990, American Society for Microbiology Bacterial Spores and Chemical Sporicidal Agents A. D. RUSSELL Welsh School of Pharmacy, University of Wales College of Cardiff, Cardiff, CFJ 3XF, Wales INTRODUCTION................................................. 99 THE BACTERIAL SPORE ................................................. 99 SPOROSTATIC AND SPORICIDAL ACTIVITY .................................................. 100 Group A: Sporostatic Compounds ................................................. 101 Phenols and cresols................................................. 101 Organic acids and esters ................................................. 101 Alcohols ................................................. 101 QACs ................................................. 101 Biguanides ................................................. 101 Organomercury compounds................................................. 101 Group B: Sporicidal Compounds ................................................. 102 Glutaraldehyde ................................................. 102 Formaldehyde ................................................. 102 Other aldehydes.................................................. 103 Chlorine-releasing agents .................................................. 103 Iodine and iodophors .................................................. 103 Peroxygens .................................................. 103 Ethylene oxide ................................................. 104 P-Propiolactone ................................................. 104 Other gases................................................. 105 RECOVERY AND REVIVAL OF INJURED SPORES ................................................. 105 SPOROGENESIS, SUSCEPTIBILITY, AND RESISTANCE .................................................. 106 Sporulation ................................................. 107 Germination................................................. 108 Outgrowth ................................................. 110 OVERCOMING SPORE RESISTANCE ................................................. 110 MECHANISMS OF SPORICIDAL ACTION..................................................111 MEDICAL AND OTHER USES OF CHEMICAL SPORICIDES ................................................. 112 Sporicidal Agents ................................................. 112 Inhibitors of Germination and Outgrowth ................................................. 113 CONCLUSIONS ................................................. 113 LITERATURE CITED................................................. 114 INTRODUCTION learning more about the ways in which sporicides act or spores resist their action, and due attention will be paid to Bacterial spores are highly resistant to chemical and these aspects. Finally, the clinical uses of sporicidal agents physical agents (25, 88, 89, 91, 102, 139, 158, 166, 171-174, will be discussed. 178, 207, 208, 224, 225). Processes designed to achieve General aspects of disinfection and disinfectants are to be sterilization of food, pharmaceutical, medical, and other found in references 73, 101, and 183. These include details, products have thus, of necessity, had to take this high level when relevant, of sporicidal activity. Spore resistance is of resistance into account. Spores are also of importance in described by Russell et al. (177) and Gardner and Peel (72). other contexts, notably, (i) as food-poisoning agents (Clos- In the United States, commercially available disinfectants tridium botulinum, C. perfringens, and Bacillus cereus), (ii) are regulated by the Environmental Protection Agency and as etiological agents (C. perfringens and C. tetani) in some must be used according to the directions specified on their infections, and (iii) as sources of antibiotics, toxins, and labels. Workers elsewhere should be familiar with regula- insecticides. Add to these the complex and fascinating series tions pertaining to their own country. of events that take place during sporulation, germination, and outgrowth and the stage is set for a comprehensive study encompassing many scientific and medical disciplines, sev- THE BACTERIAL SPORE eral of which are outside the scope of the present paper. This paper will deal with chemical sporicidal agents of the The most important sporeformers are members of the disinfectant type. Such chemicals are comparatively few in genera Bacillus and Clostridium. Certain other bacteria, number and their activity is often susceptible to environmen- e.g., Sporosarcinae, Desulfomaculum, and Sporolactobacil- tal conditions, at least some of which can be readily con- lus spp. (52), can also form spores, but will not be considered trolled. Other agents that are bactericidal and sporostatic but here. True endospores are also produced by thermophilic not usually sporicidal will also be considered when relevant. actinomycetes. Thermoactinomyces vulgaris spores are More effective sporicidal action will only be achieved by highly refractile, do not take up simple stains, have a typical 99 100 RUSSELL CLIN. MICROBIOL. REV. TABLE 2. Agents with bactericidal, sporostatic, and sporicidal activity Bactericidal agents Bactericidal agents Comment that are sporostatic that are sporicidal Group A Phenols None in group A Even high concentra- Organic acids and tions for prolonged estersa periods at ambient QACs temp are not spori- Biguanides cidal; may be Organomercurials sporicidal at ele- Alcohols vated temperatures Group B Glutaraldehyde All in group B Low concentrations Formaldehyde are sporostatic; Iodine compounds usually much higher concentra- Chlorine compounds tionertons are needed CX Hydrogen peroxide for sporicidal effect FIG. 1. "Typical" bacterial spore. The exosporium is present in Peroxy acids some, but not all, types of spores. EXO, Exosporium; OSC, outer Ethylene oxide spore coat; ISC, inner spore coat; CX, cortex-; GCW, germ cell wall; 13-Propiolactone PM, plasma membrane. a For example, the parabens [methyl, ethyl, propyl, and butyl esters of para-(4)-hydroxybenzoic acid]. spore structure, contain dipicolinic acid, and are heat resis- tant (173). (cortical membrane, germ cell wall, primordial cell wall) of The structure of a so-called typical bacterial spore is the cortex develops into the cell wall of the emergent cell depicted in Fig. 1. It is clear that the spore is a complex when the cortex is degraded during germination. entity, being composed of several different layers, some of Two membranes, the inner and outer forespore mem- which are implicated in their greater resistance than vegeta- branes, surround the forespore during germination. The tive cells to chemical or physical processes. The molecular inner forespore membrane eventually becomes the cytoplas- structure of the bacterial spore is considered in detail by mic membrane of the germinating spore, whereas the outer Ellar (57) and Warth (233). The germ cell (protoplast or core) forespore membrane persists in the spore integuments. and germ cell wall are surrounded by the cortex, external to The spore coats make up a major portion of the spore which are the inner and denser outer spore coats. An (139), consisting mainly of protein with smaller amounts of exosporium is present in some spores, but may surround just complex carbohydrates and lipid and possibly large amounts one dense spore coat. of phosphorus. The outer spore coat contains the alkali- In terms of its macromolecular constituents (Table 1), the resistant protein fraction and is associated with the presence protoplast is the location of RNA, DNA, dipicolinic acid, ofdisulfide-rich bonds. The alkali-soluble fraction is found in and most of the calcium, potassium, manganese, and phos- the inner spore coats and consists predominantly of acidic phorus present in the spore. Also present is a substantial polypeptides which can be dissociated to their unit compo- amount of low-molecular-weight basic proteins which are nents by treatment with sodium dodecyl sulfate. rapidly degraded during germination (187). From this brief consideration of the structure and compo- The cortex consists largely of peptidoglycan, some 45 to sition of the bacterial spore, it is obvious that several sites 60% of the muramic acid residues not having either a peptide exist for attack by biocides and equally obvious that the or an N-acetyl substituent but instead forming an internal spore can possess barriers which limit biocide penetration. It amide, muramic lactam (233). Peptidoglycan is the site of is the purpose of this review not only to describe the activity, action of lysozyme and of nitrous acid. A dense inner layer properties, and uses of sporicidal agents but also to consider their mechanism of action, insofar as this is known, how resistance may be presented by the spore, and how this may TABLE 1. Chemical composition of bacterial spores be overcome. Composition component Comment SPOROSTATIC AND SPORICIDAL ACTIVITY Outer spore Mainly protein Alkali resistant; removed coat by disulfide bond-re- Comparatively few antibacterial agents are actively spori- ducing agents cidal (101, 173, 180). Even quite powerful bactericides may Inner spore Mainly protein Alkali soluble be inhibitory to spore germination or outgrowth or both, i.e., coat sporostatic, rather than sporicidal. Examples include phe- Cortex Mainly peptidoglycan Differs from peptidogly- nols and cresols, quaternary ammonium compounds can of vegetative cell (QACs),