INTRODUCTION

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Antiseptics and disinfectants are used extensively in hospitals and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections (277, 454). Mounting concerns over the potential for microbial contamination and infection risks in the food and general consumer markets have also led to increased use of antiseptics and disinfectants by the general public. A wide variety of active chemical agents (or "biocides") are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection, and preservation (39). Despite this, less is known about the mode of action of these active agents than about antibiotics. In general, biocides have a broader spectrum of activity than antibiotics, and, while antibiotics tend to have specific intracellular targets, biocides may have multiple targets. The widespread use of antiseptic and disinfectant products has prompted some speculation on the development of microbial resistance, in particular cross-resistance to antibiotics. This review considers what is known about the mode of action of, and mechanisms of microbial resistance to, antiseptics and disinfectants and attempts, wherever possible, to relate current knowledge to the clinical environment.

A summary of the various types of biocides used in antiseptics and disinfectants, their chemical structures, and their clinical uses is shown in Table 1. It is important to note that many of these biocides may be used singly or in combination in a variety of products which vary considerably in activity against microorganisms. Antimicrobial activity can be influenced by many factors such as formulation effects, presence of an organic load, synergy, temperature, dilution, and test method. These issues are beyond the scope of this review and are discussed elsewhere (123, 425, 444, 446, 451).

Sterilization refers to a physical or chemical process that completely destroys or removes all microbial life, including spores. Preservation is the prevention of multiplication of microorganisms in formulated products, including pharmaceuticals and foods. A number of biocides are also used for cleaning purposes; cleaning in these cases refers to the physical removal of foreign material from a surface (40).

MECHANISMS OF ACTION

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Introduction

Considerable progress has been made in understanding the mechanisms of the antibacterial action of antiseptics and disinfectants (215, 428, 437). By contrast, studies on their modes of action against fungi (426, 436), viruses (298, 307), and protozoa (163) have been rather sparse. Furthermore, little is known about the means whereby these agents inactivate prions (503).

Whatever the type of microbial cell (or entity), it is probable that there is a common sequence of events. This can be envisaged as interaction of the antiseptic or disinfectant with the cell surface followed by penetration into the cell and action at the target site(s). The nature and composition of the surface vary from one cell type (or entity) to another but can also alter as a result of changes in the environment (57, 59). Interaction at the cell surface can produce a significant effect on viability (e.g. with glutaraldehyde) (374, 421), but most antimicrobial agents appear to be active intracellularly (428, 451). The outermost layers of microbial cells can thus have a significant effect on their susceptibility (or insusceptibility) to antiseptics and disinfectants; it is disappointing how little is known about the passage of these antimicrobial agents into different types of microorganisms. Potentiation of activity of most biocides may be achieved by the use of various additives, as shown in later parts of this review.

In this section, the mechanisms of antimicrobial action of a range of chemical agents that are used as antiseptics or disinfectants or both are discussed. Different types of microorganisms are considered, and similarities or differences in the nature of the effect are emphasized. The mechanisms of action are summarized in Table 2.

A battery of techniques are available for studying the mechanisms of action of antiseptics and disinfectants on microorganisms, especially bacteria (448). These include examination of uptake (215, 428, 459), lysis and leakage of intracellular constituents (122), perturbation of cell homeostasis (266, 445), effects on model membranes (170), inhibition of enzymes, electron transport, and oxidative phosphorylation (162, 272), interaction with macromolecules (448, 523), effects on macromolecular biosynthetic processes (133), and microscopic examination of biocide-exposed cells (35). Additional and useful information can be obtained by calculating concentration exponents (n values [219, 489]) and relating these to membrane activity (219). Many of these procedures are valuable for detecting and evaluating antiseptics or disinfectants used in combination (146, 147, 202, 210).

Similar techniques have been used to study the activity of antiseptics and disinfectants against fungi, in particular yeasts. Additionally, studies on cell wall porosity (117-119) may provide useful information about intracellular entry of disinfectants and antiseptics (204-208).

Mechanisms of antiprotozoal action have not been widely investigated. One reason for this is the difficulty in culturing some protozoa (e.g., Cryptosporidium) under laboratory conditions. However, the different life stages (trophozoites and cysts) do provide a fascinating example of the problem of how changes in cytology and physiology can modify responses to antiseptics and disinfectants. Khunkitti et al. (251-255) have explored this aspect by using indices of viability, leakage, uptake, and electron microscopy as experimental tools.

Some of these procedures can also be modified for studying effects on viruses and phages (e.g., uptake to whole cells and viral or phage components, effects on nucleic acids and proteins, and electron microscopy) (401). Viral targets are predominantly the viral envelope (if present), derived from the host cell cytoplasmic or nuclear membrane; the capsid, which is responsible for the shape of virus particles and for the protection of viral nucleic acid; and the viral genome. Release of an intact viral nucleic acid into the environment following capsid destruction is of potential concern since some nucleic acids are infective when liberated from the capsid (317), an aspect that must be considered in viral disinfection. Important considerations in viral inactivation are dealt with by Klein and Deforest (259) and Prince et al. (384), while an earlier paper by Grossgebauer is highly recommended (189).

Although several alcohols have been shown to be effective antimicrobials, ethyl alcohol (ethanol, alcohol), isopropyl alcohol (isopropanol, propan-2-ol) and n-propanol (in particular in Europe) are the most widely used (337). Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria), viruses, and fungi but are not sporicidal. They are, however, known to inhibit sporulation and spore germination (545), but this effect is reversible (513). Because of the lack of sporicidal activity, alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis. Lower concentrations may also be used as preservatives and to potentiate the activity of other biocides. Many alcohol products include low levels of other biocides (in particular chlorhexidine), which remain on the skin following evaporation of the alcohol, or excipients (including emollients), which decrease the evaporation time of the alcohol and can significantly increase product efficacy (68). In general, isopropyl alcohol is considered slightly more efficacious against bacteria (95) and ethyl alcohol is more potent against viruses (259); however, this is dependent on the concentrations of both the active agent and the test microorganism. For example, isopropyl alcohol has greater lipophilic properties than ethyl alcohol and is less active against hydrophilic viruses (e.g., poliovirus) (259). Generally, the antimicrobial activity of alcohols is significantly lower at concentrations below 50% and is optimal in the 60 to 90% range.

Little is known about the specific mode of action of alcohols, but based on the increased efficacy in the presence of water, it is generally believed that they cause membrane damage and rapid denaturation of proteins, with subsequent interference with metabolism and cell lysis (278, 337). This is supported by specific reports of denaturation of Escherichia coli dehydrogenases (499) and an increased lag phase in Enterobacter aerogenes, speculated to be due to inhibition of metabolism required for rapid cell division (101).

Glutaraldehyde. Glutaraldehyde is an important dialdehyde that has found usage as a disinfectant and sterilant, in particular for low-temperature disinfection and sterilization of endoscopes and surgical equipment and as a fixative in electron icroscopy. Glutaraldehyde has a broad spectrum of activity against bacteria and their spores, fungi, and viruses, and a considerable amount of information is now available about the ways whereby these different organisms are inactivated (Tables 2 and 3). Earlier reviews of its mechanisms of action have been published (179, 182, 374, 482).

Formaldehyde. Formaldehyde (methanal, CH₂O) is a monoaldehyde that exists as a freely water-soluble gas. Formaldehyde solution (formalin) is an aqueous solution containing ca. 34 to 38% (wt/wt) CH₂O with methanol to delay polymerization. Its clinical use is generally as a disinfectant and sterilant in liquid or in combination with low-temperature steam. Formaldehyde is bactericidal, sporicidal, and virucidal, but it works more slowly than glutaraldehyde (374, 482).

Formaldehyde-releasing agents. Several formaldehyde-releasing agents have been used in the treatment of peritonitis (226, 273). They include noxythiolin (oxymethylenethiourea), tauroline (a condensate of two molecules of the aminosulponic acid taurine with three molecules of formaldehyde), hexamine (hexamethylenetetramine, methenamine), the resins melamine and urea formaldehydes, and imidazolone derivatives such as dantoin. All of these agents are claimed to be microbicidal on account of the release of formaldehyde. However, because the antibacterial activity of taurolin is greater than that of free formaldehyde, the activity of taurolin is not entirely the result of formaldehyde action (247).

o-Phthalaldehyde. OPA is a new type of disinfectant that is claimed to have potent bactericidal and sporicidal activity and has been suggested as a replacement for glutaraldehyde in endoscope disinfection (7). OPA is an aromatic compound with two aldehyde groups. To date, the mechanism of its antimicrobial action has been little studied, but preliminary evidence (526) suggests an action similar to that of glutaraldehyde. Further investigations are needed to corroborate this opinion.

The anilides have been investigated primarily for use as antiseptics, but they are rarely used in the clinic. Triclocarban (TCC; 3,4,4'-triclorocarbanilide) is the most extensively studied in this series and is used mostly in consumer soaps and deodorants. TCC is particularly active against gram-positive bacteria but significantly less active against gram-negative bacteria and fungi (30) and lacks appreciable substantivity (persistency) for the skin (37). The anilides are thought to act by adsorbing to and destroying the semipermeable character of the cytoplasmic membrane, leading to cell death (194).

Chlorhexidine. Chlorhexidine is probably the most widely used biocide in antiseptic products, in particular in handwashing and oral products but also as a disinfectant and preservative. This is due in particular to its broad-spectrum efficacy, substantivity for the skin, and low irritation. Of note, irritability has been described and in many cases may be product specific (167, 403). Despite the advantages of chlorhexidine, its activity is pH dependent and is greatly reduced in the presence of organic matter (430). A considerable amount of research has been undertaken on the mechanism of the antimicrobial action of this important bisbiguanide (389) (Tables 2 and 4), although most of the attention has been devoted to the way in which it inactivates nonsporulating bacteria (215, 428, 430, 431, 451). Nevertheless, sufficient data are now available to examine its sporostatic and mycobacteriostatic action, its effects on yeasts and protozoa, and its antiviral activity.

Alexidine. Alexidine differs chemically from chlorhexidine in possessing ethylhexyl end groups. Alexidine is more rapidly bactericidal and produces a significantly faster alteration in bactericidal permeability (79, 80). Studies with mixed-lipid and pure phospholipid vesicles demonstrate that, unlike chlorhexidine, alexidine produces lipid phase separation and domain formation (Table 2). It has been proposed (80) that the nature of the ethylhexyl end group in alexidine, as opposed to the chlorophenol one in chlorhexidine, might influence the ability of a biguanide to produce lipid domains in the cytoplasmic membrane.

Polymeric biguanides. Vantocil is a heterodisperse mixture of polyhexamethylene biguanides (PHMB) with a molecular weight of approximately 3,000. Polymeric biguanides have found use as general disinfecting agents in the food industry and, very successfully, for the disinfection of swimming pools. Vantocil is active against gram-positive and gram-negative bacteria, although P. aeruginosa and Proteus vulgaris are less sensitive. Vantocil is not sporicidal. PHMB is a membrane-active agent that also impairs the integrity of the outer membrane of gram-negative bacteria, although the membrane may also act as a permeability barrier (64, 172). Activity of PHMB increases on a weight basis with increasing levels of polymerization, which has been linked to enhanced inner membrane perturbation (173, 174).

The diamidines are characterized chemically as described in Table 1. The isethionate salts of two compounds, propamidine (4,4-diaminodiphenoxypropane) and dibromopropamidine (2,2-dibromo-4,4-diamidinodiphenoxypropane), have been used as antibacterial agents. Their antibacterial properties and uses were reviewed by Hugo (213) and Hugo and Russell (226). Clinically, diamidines are used for the topical treatment of wounds.

The exact mechanism of action of diamidines is unknown, but they have been shown to inhibit oxygen uptake and induce leakage of amino acids (Table 2), as would be expected if they are considered as cationic surface-active agents. Damage to the cell surface of P. aeruginosa and Enterobacter cloacae has been described (400).

Chlorine-releasing agents. Excellent reviews that deal with the chemical, physical, and microbiological properties of chlorine-releasing agents (CRAs) are available (42, 130). The most important types of CRAs are sodium hypochlorite, chlorine dioxide, and the N-chloro compounds such as sodium dichloroisocyanurate (NaDCC), with chloramine-T being used to some extent. Sodium hypochlorite solutions are widely used for hard-surface disinfection (household bleach) and can be used for disinfecting spillages of blood containing human immunodeficiency virus or HBV. NaDCC can also be used for this purpose and has the advantages of providing a higher concentration of available chlorine and being less susceptible to inactivation by organic matter. In water, sodium hypochlorite ionizes to produce Na⁺ and the hypochlorite ion, OCl, which establishes an equilibrium with hypochlorous acid, HOCl (42). Between pH 4 and 7, chlorine exists predominantly as HClO, the active moiety, whereas above pH9, OCl predominates. Although CRAs have been predominantly used as hard-surface disinfectants, novel acidified sodium chlorite (a two-component system of sodium chlorite and mandelic acid) has been described as an effective antiseptic (248).

Iodine and iodophors. Although less reactive than chlorine, iodine is rapidly bactericidal, fungicidal, tuberculocidal, virucidal, and sporicidal (184). Although aqueous or alcoholic (tincture) solutions of iodine have been used for 150 years as antiseptics, they are associated with irritation and excessive staining. In addition, aqueous solutions are generally unstable; in solution, at least seven iodine species are present in a complex equilibrium, with molecular iodine (I₂) being primarily responsible for antimictrobial efficacy (184). These problems were overcome by the development of iodophors ("iodine carriers" or "iodine-releasing agents"); the most widely used are povidone-iodine and poloxamer-iodine in both antiseptics and disinfectants. Iodophors are complexes of iodine and a solubilizing agent or carrier, which acts as a reservoir of the active "free" iodine (184). Although germicidal activity is maintained, iodophors are considered less active against certain fungi and spores than are tinctures (454).

In one form or another, silver and its compounds have long been used as antimicrobial agents (55, 443). The most important silver compound currently in use is silver sulfadiazine (AgSD), although silver metal, silver acetate, silver nitrate, and silver protein, all of which have antimicrobial properties, are listed in Martindale, The Extra Pharmacopoeia (312). In recent years, silver compounds have been used to prevent the infection of burns and some eye infections and to destroy warts.

Silver nitrate. The mechanism of the antimicrobial action of silver ions is closely related to their interaction with thiol (sulfydryl, -SH) groups (32, 49, 161, 164), although other target sites remain a possibility (397, 509). Liau et al (287) demonstrated that amino acids such as cysteine and other compounds such as sodium thioglycolate containing thiol groups neutralized the activity of silver nitrate against P. aeruginosa. By contrast, amino acids containing disulfide (SS) bonds, non-sulfur-containing amino acids, and sulfur-containing compounds such as cystathione, cysteic acid, L-methionine, taurine, sodium bisulfite, and sodium thiosulfate were all unable to neutralize Ag⁺ activity. These and other findings imply that interaction of Ag⁺ with thiol groups in enzymes and proteins plays an essential role in bacterial inactivation, although other cellular components may be involved. Hydrogen bonding, the effects of hydrogen bond-breaking agents, and the specificity of Ag⁺ for thiol groups were discussed in greater detail by Russell and Hugo (443) (Table 2). Virucidal properties might also be explained by binding to -SH groups (510).

Silver sulfadiazine. AgSD is essentially a combination of two antibacterial agents, Ag⁺ and sulfadiazine (SD). The question whether the antibacterial effect of AgSD arises predominantly from only one of the compounds or via a synergistic interaction has been posed repeatedly. AgSD has a broad spectrum of activity and, unlike silver nitrate, produces surface and membrane blebs in susceptible (but not resistant) bacteria (96). AgSD binds to cell components, including DNA (332, 404). Based on a chemical analysis, Fox (153) proposed a polymeric structure of AgSD composed of six silver atoms bonding to six SD molecules by linkage of the silver atoms to the nitrogens of the SD pyrimidine ring. Bacterial inhibition would then presumably be achieved when silver binds to sufficient base pairs in the DNA helix, thereby inhibiting transcription. Similarly, its antiphage properties have been ascribed to the fact that AgSD binds to phage DNA (154, 388). Clearly, the precise mechanism of action of AgSD has yet to be solved.

Hydrogen peroxide. Hydrogen peroxide (H₂O₂) is a widely used biocide for disinfection, sterilization, and antisepsis. It is a clear, colorless liquid that is commercially available in a variety of concentrations ranging from 3 to 90%. H₂O₂ is considered environmentally friendly, because it can rapidly degrade into the innocuous products water and oxygen. Although pure solutions are generally stable, most contain stabilizers to prevent decomposition. H₂O₂ demonstrates broad-spectrum efficacy against viruses, bacteria, yeasts, and bacterial spores (38). In general, greater activity is seen against gram-positive than gram-negative bacteria; however, the presence of catalase or other peroxidases in these organisms can increase tolerance in the presence of lower concentrations. Higher concentrations of H₂O₂ (10 to 30%) and longer contact times are required for sporicidal activity (416), although this activity is significantly increased in the gaseous phase. H₂O₂ acts as an oxidant by producing hydroxyl free radicals (OH) which attack essential cell components, including lipids, proteins, and DNA. It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted (38).

Peracetic acid. Peracetic acid (PAA) (CH₃COOOH) is considered a more potent biocide than hydrogen peroxide, being sporicidal, bactericidal, virucidal, and fungicidal at low concentrations (<0.3%) (38). PAA also decomposes to safe by-products (acetic acid and oxygen) but has the added advantages of being free from decomposition by peroxidases, unlike H₂O₂, and remaining active in the presence of organic loads (283, 308). Its main application is as a low-temperature liquid sterilant for medical devices, flexible scopes, and hemodialyzers, but it is also used as an environmental surface sterilant (100, 308).

Phenolic-type antimicrobial agents have long been used for their antiseptic, disinfectant, or preservative properties, depending on the compound. It has been known for many years (215) that, although they have often been referred to as "general protoplasmic poisons," they have membrane-active properties which also contribute to their overall activity (120) (Table 2).

Phenol induces progressive leakage of intracellular constituents, including the release of K⁺, the first index of membrane damage (273), and of radioactivity from ¹⁴C-labeled E. coli (242, 265). Pulvertaft and Lumb (386) demonstrated that low concentrations of phenols (0.032%, 320 µg/ml) and other (nonphenolic) agents lysed rapidly growing cultures of E. coli, staphylococci, and streptococci and concluded that autolytic enzymes were not involved. Srivastava and Thompson (487, 488) proposed that phenol acts only at the point of separation of pairs of daughter cells, with young bacterial cells being more sensitive than older cells to phenol.

Hugo and Bloomfield (216, 217) showed with the chlorinated bis-phenol fenticlor that there was a close relationship between bactericidal activity and leakage of 260-nm-absorbing material (leakage being induced only by bactericidal concentrations). Fentichlor affected the metabolic activities of S. aureus and E. coli (217) and produced a selective increase in permeability to protons with a consequent dissipation of the proton motive force (PMF) and an uncoupling of oxidative phosphorylation (41). Chlorocresol has a similar action (124). Coagulation of cytoplasmic constituents at higher phenol concentrations, which causes irreversible cellular damage, has been described by Hugo (215).

The phenolics possess antifungal and antiviral properties. Their antifungal action probably involves damage to the plasma membrane (436), resulting in leakage of intracellular constituents. Phenol does not affect the transduction of P. aeruginosa PAO by bacteriophage F116 (301), has no effect on phage DNA within the capsid, and has little effect on several of the phage band proteins unless treatments of 20 min or longer are used (303, 304).

The bis-phenols are hydroxy-halogenated derivatives of two phenolic groups connected by various bridges (191, 446). In general, they exhibit broad-spectrum efficacy but have little activity against P. aeruginosa and molds and are sporostatic toward bacterial spores. Triclosan and hexachlorophane are the most widely used biocides in this group, especially in antiseptic soaps and hand rinses. Both compounds have been shown to have cumulative and persistent effects on the skin (313).

Triclosan. Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether; Irgasan DP 300) exhibits particular activity against gram-positive bacteria (469, 521). Its efficacy against gram-negative bacteria and yeasts can be significantly enhanced by formulation effects. For example, triclosan in combination with EDTA caused increased permeability of the outer membrane (282). Reports have also suggested that in addition to its antibacterial properties, triclosan may have anti-inflammatory activity (25, 522). The specific mode of action of triclosan is unknown, but it has been suggested that the primary effects are on the cytoplasmic membrane. In studies with E. coli, triclosan at subinhibitory concentrations inhibited the uptake of essential nutrients, while higher, bactericidal concentrations resulted in the rapid release of cellular components and cell death (393). Studies with a divalent-ion-dependent E. coli triclosan mutant for which the triclosan MIC was 10-fold greater than that for a wild-type strain showed no significant differences in total envelope protein profiles but did show significant differences in envelope fatty acids (370). Specifically, a prominent 14:1 fatty acid was absent in the resistant strain, and there were minor differences in other fatty acid species. It was proposed that divalent ions and fatty acids may adsorb and limit the permeability of triclosan to its site of action (370). Minor changes in fatty acid profiles were recently found in both E. coli and S. aureus strains for which the triclosan MICs were elevated; however, the MBCs were not affected, suggesting, as for other phenols, that the cumulative effects on multiple targets contribute to the bactericidal activity (318, 319).

Hexachlorophene. Hexachlorophene (hexachlorophane; 2,2'-dihydroxy-3,5,6,3',5',6'-hexachlorodiphenylmethane) is another bis-phenol whose mode of action has been extensively studied. The primary action of hexachlorophene, based on studies with Bacillus megatherium, is to inhibit the membrane-bound part of the electron transport chain, and the other effects noted above are secondary ones that occur only at high concentrations (92, 158, 241, 481). It induces leakage, causes protoplast lysis, and inhibits respiration. The threshold concentration for the bactericidal activity of hexachlorphene is 10 µg/ml (dry weight), but peak leakage occurs at concentrations higher than 50 µg/ml and cytological changes occur above 30 µg/ml. Furthermore, hexachlorophene is bactericidal at 0°C despite causing little leakage at this temperature. Despite the broad-spectrum efficacy of hexachlorophene, concerns about toxicity (256), in particular in neonates, have meant that its use in antiseptic products has been limited.

Chloroxylenol (4-chloro-3,5-dimethylphenol; p-chloro-m-xylenol) is the key halophenol used in antiseptic or disinfectant formulations (66). Chloroxylenol is bactericidal, but P. aeruginosa and many molds are highly resistant (66, 432). Surprisingly, its mechanism of action has been little studied despite its widespread use over many years. Because of its phenolic nature, it would be expected to have an effect on microbial membranes.

Surface-active agents (surfactants) have two regions in their molecular structures, one a hydrocarbon, water-repellent (hydrophobic) group and the other a water-attracting (hydrophilic or polar) group. Depending on the basis of the charge or absence of ionization of the hydrophilic group, surfactants are classified into cationic, anionic, nonionic, and ampholytic (amphoteric) compounds. Of these, the cationic agents, as exemplified by quaternary ammonium compounds (QACs), are the most useful antiseptics and disinfectants (160). They are sometimes known as cationic detergents. QACs have been used for a variety of clinical purposes (e.g., preoperative disinfection of unbroken skin, application to mucous membranes, and disinfection of noncritical surfaces). In addition to having antimicrobial properties, QACs are also excellent for hard-surface cleaning and deodorization.

It has been known for many years that QACs are membrane-active agents (221) (Table 2) (i.e., with a target site predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane in yeasts) (215). Salton (460) proposed the following sequence of events with microorganisms exposed to cationic agents: (i) adsorption and penetration of the agent into the cell wall; (ii) reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization; (iii) leakage of intracellular low-molecular-weight material; (iv) degradation of proteins and nucleic acids; and (v) wall lysis caused by autolytic enzymes. There is thus a loss of structural organization and integrity of the cytoplasmic membrane in bacteria, together with other damaging effects to the bacterial cell (120).

Useful information about the selectivity of membrane action can be obtained by studying the effects of biocides on protoplasts and spheroplasts suspended in various solutes. QACs cause lysis of spheroplasts and protoplasts suspended in sucrose (107, 215, 243, 428). The cationic agents react with phospholipid components in the cytoplasmic membrane (69), thereby producing membrane distortion and protoplast lysis under osmotic stress. Isolated membranes do not undergo disaggregation on exposure to QACs, because the membrane distortion is not sufficiently drastic. The non-QAC agents TCC and trichlorosalicylanide have specific effects: TCC induces protoplast lysis in ammonium chloride by increasing Cl permeability, whereas trichlorosalicylanide induces lysis in ammonium nitrate by increasing NO₃ permeability (428). In contrast, QACs (and chlorhexidine) induce lysis of protoplasts or spheroplasts suspended in various solutes because they effect generalized, rather than specific, membrane damage.

The bacterial cytoplasmic membrane provides the mechanism whereby metabolism is linked to solute transport, flagellar movement, and the generation of ATP. Protons are extruded to the exterior of the bacterial cell during metabolism. The combined potential (concentration or osmotic effect of the proton and its electropositivity) is the PMF, which drives these ancillary activities (428). The QAC cetrimide was found (121) to have an effect on the PMF in S. aureus. At its bacteriostatic concentration, cetrimide caused the discharge of the pH component of the PMF and also produced the maximum amount of 260-nm-absorbing material.

QACs are also believed to damage the outer membrane of gram-negative bacteria, thereby promoting their own uptake. This aspect of QACs is considered below (see "Intrinsic resistance of gram-negative bacteria").

The QAC cetylpyridium chloride (CPC) induces the leakage of K⁺ and pentose material from the yeast S. cerevisiae and induces protoplast lysis as well as interacting with crude cell sap (205). Unlike chlorhexidine, however, no biphasic effect on protoplast lysis was observed. The initial toxic effect of QACs on yeast cells is a disorganization of the plasma membranes, with organized lipid structures in the membranes (and in lipid bilayers) being disrupted.

QACs are sporostatic; they inhibit the outgrowth of spores (the development of a vegetative cell from a germinated spore) but not the actual germination processes (development from dormancy to a metabolically active state), albeit by an unknown mechanism (414). Likewise, the QACs are not mycobactericidal but have a mycobacteriostatic action, although the actual effects on mycobacteria have been little studied (419).

The QACs have an effect on lipid, enveloped (including human immunodeficiency virus and HBV) but not nonenveloped viruses (394, 485, 486). QAC-based products induced disintegration and morphological changes of human HBV, resulting in loss of infectivity (382). In studies with different phages (298-301, 303-305, 307), CPC significantly inhibited transduction by bacteriophage F116 and inactivated the phage particles. Furthermore, CPC altered the protein bands of F116 but did not affect the phage DNA within the capsid.

Many heat-sensitive medical devices and surgical supplies can be effectively sterilized by liquid sterilants (in particular glutaraldehyde, PAA, and hydrogen peroxide) or by vapor-phase sterilization systems (Table 1). The most widely used active agents in these "cold" systems are ethylene oxide, formaldehyde and, more recently developed, hydrogen peroxide and PAA. Ethylene oxide and formaldehyde are both broad-spectrum alkylating agents. However, their activity is dependent on active concentration, temperature, duration of exposure, and relative humidity (87). As alkylating agents, they attack proteins, nucleic acids, and other organic compounds; both are particularly reactive with sulfhydryl and other enzyme-reactive groups. Ethylene oxide gas has the disadvantages of being mutagenic and explosive but is not generally harsh on sensitive equipment, and toxic residuals from the sterilization procedure can be routinely eliminated by correct aeration. Formaldehyde gas is similar and has the added advantage of being nonexplosive but is not widely used in health care. Vapor-phase hydrogen peroxide and PAA are considered more active (as oxidants) at lower concentrations than in the liquid form (334). Both active agents are used in combination with gas plasma in low-temperature sterilization systems (314). Their main advantages over other vapor-phase systems include low toxicity, rapid action, and activity at lower temperature; the disadvantages include limited penetrability and applications.

MECHANISMS OF RESISTANCE

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Introduction

As stated above, different types of microorganisms vary in their response to antiseptics and disinfectants. This is hardly surprising in view of their different cellular structure, composition, and physiology. Traditionally, microbial susceptibility to antiseptics and disinfectants has been classified based on these differences; with recent work, this classification can be further extended (Fig. 1). Because different types of organisms react differently, it is convenient to consider bacteria, fungi, viruses, protozoa, and prions separately.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Descending order of resistance to antiseptics and disinfectants. The asterisk indicates that the conclusions are not yet universally agreed upon.

In recent years, considerable progress has been made in understanding more fully the responses of different types of bacteria (mycobacteria, nonsporulating bacteria, and bacterial spores) to antibacterial agents (43, 84, 414, 415, 419, 422, 496). As a result, resistance can be either a natural property of an organism (intrinsic) or acquired by mutation or acquisition of plasmids (self-replicating, extrachromosomal DNA) or transposons (chromosomal or plasmid integrating, transmissible DNA cassettes). Intrinsic resistance is demonstrated by gram-negative bacteria, bacterial spores, mycobacteria, and, under certain conditions, staphylococci (Table 5). Acquired, plasmid-mediated resistance is most widely associated with mercury compounds and other metallic salts. In recent years, acquired resistance to certain other types of biocides has been observed, notably in staphylococci.

For an antiseptic or disinfectant molecule to reach its target site, the outer layers of a cell must be crossed. The nature and composition of these layers depend on the organism type and may act as a permeability barrier, in which there may be a reduced uptake (422, 428). Alternatively but less commonly, constitutively synthesized enzymes may bring about degradation of a compound (43, 214, 358). Intrinsic (innate) resistance is thus a natural, chromosomally controlled property of a bacterial cell that enables it to circumvent the action of an antiseptic or disinfectant. Gram-negative bacteria tend to be more resistant than gram-positive organisms, such as staphylococci (Table 6).

Intrinsic resistance of bacterial spores. Bacterial spores of the genera Bacillus and Clostridium have been widely studied and are invariably the most resistant of all types of bacteria to antiseptics and disinfectants (43, 46, 150, 414, 418, 420, 422, 423, 457). Although Bacillus species are generally not pathogenic, their spores are widely used as indicators of efficient sterilization. Clostridium species are significant pathogens; for example, C. difficile is the most common cause of hospital-acquired diarrhea (478). Many biocides are bactericidal or bacteristatic at low concentrations for nonsporulating bacteria, including the vegetative cells of Bacillus and Clostridium species, but high concentrations may be necessary to achieve a sporicidal effect (e.g., for glutaraldehyde and CRAs). By contrast, even high concentrations of alcohol, phenolics, QACs, and chlorhexidine lack a sporicidal effect, although this may be achieved when these compounds are used at elevated temperatures (475).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Development of resistance of Bacillus subtilis during sporulation. Roman numerals indicate the sporulation stage from III (engulfment of the forespore) to VII (release of the mature spore). Arabic numbers indicate the time (hours) following the onset of sporulation and the approximate times at which resistance develops against biocides (262). CHG, chlorhexidine; CPC, cetylpyridinium chloride; NaDCC, sodium dichloroisocyanurate.