CLINICAL MICROBIOLOGY REVIEWS, Jan. 1999, p. 147–179 0893-8512/99/$04.
00 0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 12, No. 1
Antiseptics and Disinfectants: Activity, Action, and Resistance
A. DENVER RUSSELL2
STERIS Corporation, St. Louis Operations, St. Louis, Missouri 63166,1 and Welsh School of Pharmacy, Cardiff University, Cardiff CF1 3XF, United Kingdom2 INTRODUCTION .......................................................................................................................................................148 DEFINITIONS ............................................................................................................................................................148 MECHANISMS OF ACTION ...................................................................................................................................148 Introduction .............................................................................................................................................................148 General Methodology .............................................................................................................................................148 Alcohols ....................................................................................................................................................................151 Aldehydes .................................................................................................................................................................151 Glutaraldehyde ....................................................................................................................................................151 Formaldehyde ......................................................................................................................................................153 Formaldehyde-releasing agents.........................................................................................................................153 o-Phthalaldehyde.................................................................................................................................................153 Anilides.....................................................................................................................................................................153 Biguanides................................................................................................................................................................153 Chlorhexidine ......................................................................................................................................................153 Alexidine...............................................................................................................................................................154 Polymeric biguanides..........................................................................................................................................154 Diamidines ...............................................................................................................................................................155 Halogen-Releasing Agents .....................................................................................................................................155 Chlorine-releasing agents ..................................................................................................................................155 Iodine and iodophors .........................................................................................................................................155 Silver Compounds...................................................................................................................................................155 Silver nitrate........................................................................................................................................................156 Silver sulfadiazine...............................................................................................................................................156 Peroxygens ...............................................................................................................................................................156 Hydrogen peroxide..............................................................................................................................................156 Peracetic acid ......................................................................................................................................................156 Phenols .....................................................................................................................................................................156 Bis-Phenols ..............................................................................................................................................................157 Triclosan ..............................................................................................................................................................157 Hexachlorophene.................................................................................................................................................157 Halophenols .............................................................................................................................................................157 Quaternary Ammonium Compounds ...................................................................................................................157 Vapor-Phase Sterilants ..........................................................................................................................................158 MECHANISMS OF RESISTANCE..........................................................................................................................158 Introduction .............................................................................................................................................................158 Bacterial Resistance to Antiseptics and Disinfectants ......................................................................................158 Intrinsic Bacterial Resistance Mechanisms ........................................................................................................158 Intrinsic resistance of bacterial spores............................................................................................................159 Intrinsic resistance of mycobacteria ................................................................................................................160 Intrinsic resistance of other gram-positive bacteria......................................................................................161 Intrinsic resistance of gram-negative bacteria ...............................................................................................161 Physiological (phenotypic) adaption as an intrinsic mechanism.................................................................162 Acquired Bacterial Resistance Mechanisms .......................................................................................................164 Plasmids and bacterial resistance to antiseptics and disinfectants ............................................................164 (i) Plasmid-mediated antiseptic and disinfectant resistance in gram-negative bacteria......................164 (ii) Plasmid-mediated antiseptic and disinfectant resistance in staphylococci .....................................165 (iii) Plasmid-mediated antiseptic and disinfectant resistance in other gram-positive bacteria..........166 Mutational resistance to antiseptics and disinfectants.................................................................................166 Mechanisms of Fungal Resistance to Antiseptics and Disinfectants ..............................................................167 Mechanisms of Viral Resistance to Antiseptics and Disinfectants .................................................................168 Mechanisms of Protozoal Resistance to Antiseptics and Disinfectants..........................................................169 Mechanisms of Prion Resistance to Disinfectants.............................................................................................169
* Corresponding author. Present address: STERIS Corporation, 5960 Heisley Rd., Mentor, OH 44060. Phone: (440) 354-2600. Fax: (440) 354-7038. E-mail: firstname.lastname@example.org. 147
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CONCLUSIONS .........................................................................................................................................................169 REFERENCES ............................................................................................................................................................170 INTRODUCTION 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 crossresistance 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). DEFINITIONS “Biocide” is a general term describing a chemical agent, usually broad spectrum, that inactivates microorganisms. Because biocides range in antimicrobial activity, other terms may be more speciﬁc, including “-static,” referring to agents which inhibit growth (e.g., bacteriostatic, fungistatic, and sporistatic) and “-cidal,” referring to agents which kill the target organism (e.g., sporicidal, virucidal, and bactericidal). For the purpose of this review, antibiotics are deﬁned as naturally occurring or synthetic organic substances which inhibit or destroy selective bacteria or other microorganisms, generally at low concentrations; antiseptics are biocides or products that destroy or inhibit the growth of microorganisms in or on living tissue (e.g. health care personnel handwashes and surgical scrubs); and disinfectants are similar but generally are products or biocides that are used on inanimate objects or surfaces. Disinfectants can be sporostatic but are not necessarily sporicidal. 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 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. General Methodology 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
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ANTISEPTICS AND DISINFECTANTS TABLE 1. Chemical structures and uses of biocides in antiseptics and disinfectants
Continued on following page
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
an aspect that must be considered in viral disinfection. which is responsible for the shape of virus particles and for the protection of viral nucleic acid. MICROBIOL.
. the capsid.150
MCDONNELL AND RUSSELL TABLE 1—Continued
CLIN. and the viral genome. Important considerations in viral inactivation are dealt with by Klein and Deforest (259) and Prince et al.
Continued on following page
predominantly the viral envelope (if present). derived from the host cell cytoplasmic or nuclear membrane. 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). REV.
high concentrations cause congealing of cytoplasm Induction of leakage of amino acids Phase separation and domain formation of membrane lipids Leakage. isopropyl alcohol is considered slightly
more efﬁcacious against bacteria (95) and ethyl alcohol is more potent against viruses (259). however. silver ions Halogens Peroxygens
. ethyl alcohol (ethanol. outer membrane) Cytoplasmic (inner) membrane
Glutaraldehyde EDTA. They are. but based on the increased efﬁcacy in the presence of water. alexidine Phenols
Cross-linking of proteins Gram-negative bacteria: removal of Mg2 . viruses. in particular for low-temperature disinfection and sterilization of endoscopes and surgical equipment and as a ﬁxative in electron
TABLE 2. isopropyl alcohol has greater lipophilic properties than ethyl alcohol and is less active against hydrophilic viruses (e. or excipients (including emollients). 337). Glutaraldehyde is an important dialdehyde that has found usage as a disinfectant and sterilant. alcohol). In general. speculated to be due to inhibition of metabolism required for rapid cell division (101). while an earlier paper by Grossgebauer is highly recommended (189). This is supported by speciﬁc reports of denaturation of Escherichia coli dehydrogenases (499) and an increased lag phase in Enterobacter aerogenes. Little is known about the speciﬁc mode of action of alcohols. release of some LPS Generalized membrane damage involving phospholipid bilayers Low concentrations affect membrane integrity. which remain on the skin following evaporation of the alcohol. propan-2-ol) and n-propanol (in particular in Europe) are the most widely used (337). isopropyl alcohol (isopropanol. Because of the lack of sporicidal activity. this is dependent on the concentrations of both the active agent and the test microorganism. other permeabilizers QACs Chlorhexidine Diamines PHMB. which decrease the evaporation time of the alcohol and can signiﬁcantly increase product efﬁcacy (68). Lower concentrations may also be used as preservatives and to potentiate the activity of other biocides. the antimicrobial activity of alcohols is signiﬁcantly lower at concentrations below 50% and is optimal in the 60 to 90% range. For example. Alcohols Although several alcohols have been shown to be effective antimicrobials. which oxidize thiol groups in enzymes and proteins. alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis. Summary of mechanisms of antibacterial action of antiseptics and disinfectants
Target Antiseptic or disinfectant Mechanism of action
Cell envelope (cell wall. Aldehydes Glutaraldehyde. RNA. Many alcohol products include low levels of other biocides (in particular chlorhexidine). 12. but this effect is reversible (513). however. with subsequent interference with metabolism and cell lysis (278. known to inhibit sporulation and spore germination (545). and fungi but are not sporicidal. Generally. 1999 TABLE 1—Continued
ANTISEPTICS AND DISINFECTANTS
(384). some cause uncoupling Cross-linking of proteins. poliovirus) (259). sulfoxides. and DNA Cross-linking of proteins in cell envelope and elsewhere in the cell Intercalation of an acridine molecule between two layers of base pairs in DNA Membrane-bound enzymes (interaction with thiol groups) Inhibition of DNA synthesis DNA strand breakage Oxidation of thiol groups to disulﬁdes. it is generally believed that they cause membrane damage and rapid denaturation of proteins. Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria). PAA: disruption of thiol groups in proteins and enzymes
Cross-linking of macromolecules DNA intercalation Interaction with thiol groups Effects on DNA Oxidizing agents
Formaldehyde Glutaraldehyde Acridines Silver compounds Halogens Hydrogen peroxide..VOL. or disulfoxides Hydrogen peroxide: activity due to from formation of free hydroxy radicals ( OH).g.
. possibly representing the reactive sites (65). untreated spores even when the spores are subsequently incubated in germination medium... 352)...... 508) action. speciﬁcally with unprotonated amines on the cell surface. research was undertaken to evaluate the nature of its bactericidal (339–344.... inhibition of transport in gram-negative bacteria (179). L-Alanine is considered to act by binding to a speciﬁc receptor on the spore coat.. 182. uptake of glutaraldehyde is greater during germination and outgrowth than with mature spores but still lower than with vegetative cells.... 339–341...1%) inhibit germination. It has been observed by various authors (182. this interaction reduces the release of dipicolinic acid (DPA) from heated spores and the lysis induced by mercaptoethanol (or thioglycolate)-peroxide combinations. 380) that the greater sporicidal activity of glutaraldehyde at alkaline pH is not reﬂected by differences in uptake. These bactericidal studies demonstrated (374) a strong binding of glutaraldehyde to outer layers of organisms such as E. subtilis spores.. 414). Glutaraldehyde is also mycobactericidal.. Novel glutaraldehyde formulations based on acidic rather than alkaline glutaraldehyde.1 to 1%) signiﬁcantly reduce the uptake of L-alanine. Mechanism of antimicrobial action of glutaraldehyde
Target microorganism Glutaraldehyde action
Bacterial spores . inhibition of spheroplast and protoplast lysis in hypotonic media (340... coli and Staphylococcus aureus (179. During sporulation. albeit by an unknown mechanism (379... 316). again indicating an effect at the outermost regions of the cell.. S. more reactive sites will be formed at the cell surface..Mechanism of action not known
icroscopy. Low concentrations of the dialdehyde (0... Strong interaction of glutaraldehyde with lysine and other amino acids has been demonstrated (450).... It reduces the activity of hepatitis B surface antigen (HBsAg) and especially hepatitis B core antigen ([HBcAg] in hepatitis B virus [HBV]) (3) and interacts with lysine residues on the surface of hepatitis A virus (HAV) (362). they are committed irreversibly to losing their dormant properties (491). Clearly. thereby destroying the ability of the spore to function solely as a result of this surface phenomenon. MICROBIOL. and inhibition of RNA. Glutaraldehyde-treated spores retain their refractivity.. suggests an additional effect on the inner membrane.. the cell eventually becomes less susceptible to glutaraldehyde (see “Intrinsic resistance of bacterial spores”).. It is conceivable that acidic glutaraldehyde interacts with and remains at the cell surface whereas alkaline glutaraldehyde penetrates more deeply into the spore.. the mechanism of action of glutaraldehyde involves a strong association with the outer layers of bacterial cells... having the same appearance under the phase-contrast microscope as normal. Earlier work had suggested that the fungal cell wall was a major target site (179.... 344).. DNA.. which is analogous to the peptidoglycan found in bacterial cell walls. By contrast. The bacterial spore presents several sites at which interaction with glutaraldehyde is possible. REV. whereas much higher concentrations (2%) are sporicidal. Subsequently. aureus (453) and of sodium lauryl sulfate-induced lysis in E. Low concentrations
. 182. possibly as a result of a sealing effect of the aldehyde on the cell surface. 374.. Unfortunately. coli (340. 181.... prevention of lysostaphin-induced lysis in S. 344)..... The aldehyde. inhibition of transport processes into cell Fungi ... 378). 376.... if not all. 344).... interacts strongly with the outer spore layers (508. no critical studies have as yet been undertaken to evaluate the nature of this action (419).. 507... Glutaraldehyde is normally used as a 2% solution to achieve a sporicidal effect (16.. fungi.Fungal cell wall appears to be a primary target site. Germination may be deﬁned as an irreversible process in which there is a change of an activated spore from a dormant to a metabolically active state within a short period...... 374. This contention is at odds with the hypothesis of Bruch (65). subtilis (377. 212. leading to the production of spheroplasts or protoplasts and the subsequent prevention of lysis by glutaraldehyde when these forms are diluted in a hypotonic environment. but involve proteinDNA cross-links and capsid changes Protozoa .. but probably involves mycobacterial cell wall Other nonsporulating bacteria. E.. and viruses. By contrast. 482).. Glutaraldehyde is a potent virucidal agent (143. low concentrations ( 0.... germinating and outgrowing cells reacquire sensitivity. high concentrations are sporicidal. Earlier reviews of its mechanisms of action have been published (179. who envisaged the acidic form penetrating the coat and reacting with the cortex while the alkaline form attacked the coat.. a ﬁnding substantiated by the fact that the dialdehyde prevents the selective release of some membrane-bound enzymes of Micrococcus lysodeikticus (138). especially the major wall component. with postulated interaction with chitin Viruses .
TABLE 3. chitin.. Mechanisms involved in the revival of glutaraldehyde-treated spores are discussed below (see “Intrinsic resistance of bacterial spores”). 260)... 344) and of periplasmic enzymes (179).. As the external pH is altered from acidic to alkaline. cross-linking of amino groups in protein.. at both acidic and alkaline pHs.1%) prevent phase darkening of spores and also prevent the decrease in optical density associated with a late event in germination. Low concentrations of both acidic and alkaline glutaraldehyde increase the surface hydrophobicity of spores..Strong association with outer layers of gram-positive and gram-negative bacteria. leading to a more rapid bactericidal effect..Action unknown.. There is. The improved sporicidal activity claimed for these products may be obtained by agents that potentiate the activity of the dialdehyde (414. The cross-links thus obtained mean that the cell is then unable to undertake most. and vegetative cells of Bacillus subtilis bind more glutaraldehyde than do rest-
ing spores of B.. uptake per se reﬂects binding and not necessarily penetration into the spore. Partial or entire removal of the cell wall in hypertonic medium. however.. higher concentrations (0. inhibition of dehydrogenase activity (343. Glutaraldehyde is more active at alkaline than at acidic pHs... no evidence to support this theory. although interaction with a particular site does not necessarily mean that this is associated with spore inactivation.. which beneﬁt from the greater inherent stability of the aldehyde at lower pH.. The ﬁrst reports in 1964 and 1965 (182) demonstrated that glutaraldehyde possessed high antimicrobial activity.152
MCDONNELL AND RUSSELL
CLIN......Low concentrations inhibit germination. have been produced... 450) and sporicidal (180.... 343. Glutaraldehyde at high concentrations inhibits the uptake of L-[14C]alanine by B. There are no recent studies of the mechanisms of fungicidal action of glutaraldehyde.Actual mechanisms unknown.. of its essential functions. and a considerable amount of information is now available about the ways whereby these different organisms are inactivated (Tables 2 and 3).. probably as a consequence of strong interaction with outer cell layers Mycobacteria. where access of substrate to enzyme is prohibited.. and once spores are triggered to germinate. 509).. Glutaraldehyde has a broad spectrum of activity against bacteria and their spores. as yet.. Such an effect could explain its inhibitory action on transport and on enzyme systems. coli.. Glutaraldehyde exerts an early effect on the germination process..... 421).. and protein synthesis (320). aureus.
lipid-enveloped viruses more sensitive than nonenveloped viruses... and imidazolone derivatives such as dantoin.. high concentrations cause intracellular coagulation Viruses . 442) that interacts with protein (156.. furfural.. Low concentrations of formaldehyde are sporostatic and inhibit germination (512). 1999
ANTISEPTICS AND DISINFECTANTS
( 0. Formaldehyde (methanal. aldehydes (i...g. sulfhydryl. in particular in handwashing and oral products but also as a disinfectant and preservative.. which were postulated to be involved in the ejection of the nucleic acid. less toward cysts
tauroline (a condensate of two molecules of the aminosulponic acid taurine with three molecules of formaldehyde).... In light of this. As pointed out above. 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). irritability has been described and in many cases may be product speciﬁc (167..Not sporicidal but prevents development of spores. OPA is an aromatic compound with two aldehyde groups. Most of the other aldehydes (glutaraldehyde... Biguanides Chlorhexidine.. castellanii demonstrate membrane activity (leakage) toward trophozoites..... although phenol groups bind little formaldehyde (155). The anilides are thought to act by adsorbing to and destroying the semipermeable character of the cytoplasmic membrane...4.. the mechanism of its antimicrobial action has been little studied. the resins melamine and urea formaldehydes.. inhibits spore outgrowth but not germination Mycobacteria. Concentrations of glutaraldehyde greater than 0.... Formaldehyde also reacts extensively with nucleic acid (489) (e... Glutaraldehyde and other aldehydes were tested for their ability to form protein-DNA cross-links in simian virus 40 (SV40)..... but it works more slowly than glutaraldehyde (374.. thereby inhibiting DNA synthesis (369). 157).. but preliminary evidence (526) suggests an action similar to that of glutaraldehyde. because the antibacterial activity of taurolin is greater than that of free formaldehyde... implying that the phage genome had been ejected..e...2 and is only slowly inactivated at pH 8..Mycobacteristatic (mechanism unknown) but not mycobactericidal Other nonsporulating bacteria. Formaldehyde is an extremely reactive chemical (374. However. Further investigations are needed to corroborate this opinion. which formed such cross-links (144. In other words. Formaldehyde-releasing agents. This is due in particular to its broad-spectrum efﬁcacy..3 (21).25% signiﬁcantly affected the transduction of this phage... the complete poliovirus particle is much more sensitive than poliovirus RNA. Formaldehyde alters HBsAg and HBcAg of HBV (3)....05 and 0.. 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).. All of these agents are claimed to be microbicidal on account of the release of formaldehyde. 273). its activity is pH dependent and is greatly reduced in the presence of organic matter (430). CH2O) is a monoaldehyde that exists as a freely water-soluble gas. causing protoplast and spheroplast lysis. DNA (155). Chlorhexidine is probably the most widely used biocide in antiseptic products. perhaps the lipid moieties Protozoa .... but they are rarely used in the clinic...Membrane-active agent. Despite the advantages of chlorhexidine. and hydroxyl groups (371).... and o-phthalaldehyde [OPA]) that have sporicidal activity are dialdehydes (and of these.. o-Phthalaldehyde... A considerable amount of research has been undertaken on the mechanism of the antimicrobial action of this important bisbiguanide (389) (Tables 2 and 4).. It has long been considered to be sporicidal by virtue of its ability to penetrate into the interior of bacterial spores (500). Formaldehyde solution (formalin) is an aqueous solution containing ca.. 307). glyoxyl. and benzylaldehyde) without detectable cross-linking ability had no effect on SV40 DNA synthesis. glutaraldehyde also interacted with phage F116 proteins. 297). the transduction process was more sensitive to the aldehyde than was the phage itself.. Many glutaraldehyde-treated P. substantivity for the skin. hexamine (hexamethylenetetramine. Anilides The anilides have been investigated primarily for use as antiseptics.. the activity of taurolin is not entirely the result of formaldehyde action (247). and RNA (155) in vitro. 403). the differences in sensitivity probably reﬂect major structural variations in the two viruses (75)... The interaction with protein results from a combination with the primary amide as well as with the amino groups.Recent studies against A.. Mechanisms of antimicrobial action of chlorhexidine
Type of microorganism Chlorhexidine action
Bacterial spores . The aldehyde was possibly bound to F116 double-stranded DNA but without affecting the molecule. and formaldehyde...4 -triclorocarbanilide) is the most extensively studied in this series and is used mostly in consumer soaps and deodorants.. succinaldehyde. effect possibly on viral envelope. respectively...005% interacts with the capsid proteins of poliovirus and echovirus. leading to cell death (194).. and low irritation. 12. it forms protein-DNA cross-links in SV40... and cross-linking properties must play a considerable role in this activity. It is difﬁcult to pinpoint accurately the mechanism(s) responsible for formaldehyde-induced microbial inactivation. 34 to 38% (wt/wt) CH2O with methanol to delay polymerization. To date. 306.. Of note... Several formaldehyde-releasing agents have been used in the treatment of peritonitis (226. Formaldehyde. its interactive. and virucidal. Clearly. glyoxal. The distance between the two aldehyde groups in glutaraldehyde (and possibly in OPA) may be optimal for interaction of these-CHO groups in nucleic acids and especially in proteins and enzymes (428). it has been inferred that glutaraldehyde-induced loss of infectivity is associated with capsid changes (21)... high concentrations cause precipitation of proteins and nucleic acids Yeasts... causing protoplast lysis and intracellular leakage.1%) of alkaline glutaraldehyde are effective against puriﬁed poliovirus. They include noxythiolin (oxymethylenethiourea)... prionaldehyde.. inhibited DNA synthesis (369).. although most of the attention has been devoted to the way in which it
. glyoxyl and succinaldehyde are weakly active)... glutaraldehyde.Membrane-active agent......Low activity against many viruses. methenamine)..1 to 0. Formaldehyde is bactericidal.. the DNA of bacteriophage T2) (190). sporicidal.. aeruginosa F116 phage particles had empty heads.. Glutaraldehyde at the low concentrations of 0. acetaldehyde... whereas acrolein.VOL.. Bacteriophages were recently studied to obtain information about mechanisms of virucidal action (298–304... 271..
TABLE 4... Its clinical use is generally as a disinfectant and sterilant in liquid or in combination with low-temperature steam.. Triclocarban (TCC.... whereas poliovirus RNA is highly resistant to aldehyde concentrations up to 1% at pH 7. 3. It has been proposed that formaldehyde acts as a mutagenic agent (291) and as an alkylating agent by reaction with carboxyl.. 482).
Permeability changes ensue. Alexidine differs chemically from chlorhexidine in possessing ethylhexyl end groups. Binding sites could thus be reduced in number or masked in germinating cells. 80). Little is known about the uptake of chlorhexidine by bacterial spores. High concentrations also failed to inactivate Pseudomonas aeruginosa phage F116 and had no effect on phage DNA within the capsid or on phage proteins (301). or poliovirus (34). unlike chlorhexidine. This substantiated an earlier ﬁnding (306) that chlorhexidine bound poorly to F116 particles. 173. Chlorhexidine is a bactericidal agent (120. Polymeric biguanides have found use as general disinfecting agents in the food industry and. might inﬂuence the ability of a biguanide to produce lipid domains in the cytoplasmic membrane. for the disinfection of swimming pools. The agent then crosses the cell wall or outer membrane.000. be reﬂected in the relative uptake of chlorhexidine. it is membrane disruption rather than ATPase inactivation that is associated with its lethal effects (24. and there is believed to be an altered function of some membrane-associated enzymes. 430. but leakage is reduced at higher biocide concentrations because of the coagulation of the cytosol. PHMB is a membrane-active agent that also impairs the integrity of the outer membrane of gram-negative bacteria. Mycobacteria are generally highly resistant to chlorhexidine (419). The proposed sequence of events during its interaction with the cell envelope of E. sufﬁcient data are now available to examine its sporostatic and mycobacteriostatic action. the inhibitory effects of chlorhexidine on mycobacteria may not be dissimilar to those on susceptible bacteria. The reason for its lack of effect on the former process but its signiﬁcant activity against the latter is unclear. It could. very successfully. Damage to the delicate semipermeable membrane is followed by leakage of intracellular constituents. Work to date suggests that chlorhexidine has a similar effect on the trophozoites of Acanthameoba castellanii. Little is known about the uptake of chlorhexidine (and other antiseptics and disinfectants) by mycobacteria and on the biochemical changes that occur in the treated cells. with a maximum effect occurring within 20 s. the cytoplasm becomes congealed.
Chlorhexidine is not sporicidal (discussed in “Mechanisms of resistance”). 527) demonstrate an effect on the fungal plasma membrane but with signiﬁcant actions elsewhere in the cell (47). 474). although P. Chlorhexidine does not inactivate nonenveloped viruses such as rotavirus (485). 136. Chlorhexidine has little effect on the germination of bacterial spores (414. Mycobacterium avium-intracellulare is considerably more resistant than other mycobacteria (48). who found that the uptake of chlorhexidine by E. Damage to the outer cell layers takes place (139) but is insufﬁcient to induce lysis or cell death. with the cysts being less sensitive (251–255). Although chlorhexidine collapses the membrane potential. 272). presumably by passive diffusion. However. although this has yet to be shown experimentally. The antiviral activity of chlorhexidine is variable. PHMB causes domain formation of the acidic phospholipids of the cytoplasmic membrane (61–64. HAV (315). as opposed to the chlorophenol one in chlorhexidine. Increasing concentrations of chlorhexidine (up to 25 g/ml) induce progressive lysis of Saccharomyces cerevisiae protoplasts. 137. As a result. Furthermore. so that there is a biphasic effect on membrane permeability. Activity of PHMB increases on a weight basis with increasing levels of polymerization. Vantocil is a heterodisperse mixture of polyhexamethylene biguanides (PHMB) with a molecular weight of approximately 3. 172. chlorhexidine “partitions” into the cell wall.
inactivates nonsporulating bacteria (215. In yeasts. 432. which can be measured by appropriate techniques. coli is as follows: (i) there is rapid attraction of
. 447) but inhibits outgrowth (447). and its activity is restricted to the lipid-enveloped viruses (361). its effects on yeasts and protozoa. in whole cells. but higher biguanide concentrations result in reduced lysis (205). Chlorhexidine has a biphasic effect on protoplast lysis. Presumably. Studies with mixed-lipid and pure phospholipid vesicles demonstrate that. 172). and its antiviral activity. Alexidine is more rapidly bactericidal and produces a signiﬁcantly faster alteration in bactericidal permeability (79. aureus was very rapid and depended on the chlorhexidine concentration and pH. 227). by using [14C]chlorhexidine gluconate. with a consequent reduction in leakage (222–224. and subsequently attacks the bacterial cytoplasmic or inner membrane or the yeast plasma membrane. Even high concentrations of the bisbiguanide do not affect the viability of Bacillus spores at ambient temperatures (473. Its activity was found by Ranganathan (389) to be restricted to the nucleic acid core or the outer coat. only high biguanide concentrations inhibit membrane-bound ATPase (83). although coatless forms take up more of the compound than do “normal” spores (474). (199) to be an inhibitor of both membrane-bound and soluble ATPase as well as of net K uptake in Enterococcus faecalis. 290). It has been proposed (80) that the nature of the ethylhexyl end group in alexidine. 428. (222–224). 215). Leakage is not per se responsible for cellular inactivation but is a consequence of cell death (445). The effects of chlorhexidine on yeast cells are probably similar to those previously described for bacteria (204–207). sufﬁcient changes occur in the spore structure to permit an increased uptake of the biguanide. the yeast cell wall may have some effect in limiting the uptake of the biguanide (208). REV. 174). since germinating cells take up much less of the bisbiguanide than do outgrowing forms (474). although it is likely that the latter would be a more important target site. Polymeric biguanides. and cytoplasm of cells (205). although the membrane may also act as a permeability barrier (64. Alexidine. which suggests that the enzyme is not a primary target for chlorhexidine action. The ﬁndings presented here and elsewhere (47. alexidine produces lipid phase separation and domain formation (Table 2). however. aeruginosa and Proteus vulgaris are less sensitive. the uptake by bacteria (145) and yeasts (204) was shown to be extremely rapid. gram-positive cocci (48). High concentrations of chlorhexidine cause coagulation of intracellular constituents. Its interaction and uptake by bacteria were studied initially by Hugo et al. Since the MICs for some mycobacteria are on the order of those for chlorhexidine-sensitive. Vantocil is active against gram-positive and gram-negative bacteria. 431. with reduced lysis at higher biguanide concentrations. the transduction process was more sensitive to chlorhexidine and other biocides than was the phage itself. Furr (163) reviewed the effects of chlorhexidine and other biocides on Acanthameoba and showed that membrane damage in these protozoa is a signiﬁcant factor in their inactivation. Studies with different types of bacteriophages have shown that chlorhexidine has no effect on MS2 or K coliphages (300). MICROBIOL. More recently. Nevertheless. Vantocil is not sporicidal. Unlike chlorhexidine but similar to alexidine (Table 2). Chlorhexidine was claimed by Harold et al. coli and S. which has been linked to enhanced inner membrane perturbation (173. although a marked sporicidal effect is achieved at elevated temperatures (475). plasma membrane. 422. An initial high rate of leakage rises as the concentration of chlorhexidine increases. Chlorhexidine is not always considered a particularly effective antiviral agent. 451).154
MCDONNELL AND RUSSELL
the antimicrobial action of iodine is rapid. (ii) the integrity of the outer membrane is impaired. this depends on the pH and concentration of available chlorine (408. and microbiological properties of chlorine-releasing agents (CRAs) are available (42. Iodine and iodophors. fungicidal. and fatty acids (15. 267]). chlorine dioxide. Deleterious effects of CRAs on bacterial DNA that involve
the formation of chlorinated derivatives of nucleotide bases have been described (115. and the N-chloro compounds such as sodium dichloroisocyanurate (NaDCC). coli within 5 min. further studies are needed to explain the antiviral action of CRAs. HOCl (42). 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. Excellent reviews that deal with the chemical. aeruginosa and Enterobacter cloacae has been described (400). Clearly. with an increase in inner membrane permeability (K loss) accompanied by bacteriostasis. Similarly to bacteria. they are associated with irritation and excessive staining. and sporicidal (184). with strong and speciﬁc adsorption to phosphate-containing compounds. Halogen-Releasing Agents Chlorine. 486). chlorine dioxide inhibited bacterial protein synthesis (33). which acts as a reservoir of the active “free” iodine (184).4-diaminodiphenoxypropane) and dibromopropamidine (2. Between pH 4 and 7. which establishes an equilibrium with hypochlorous acid. 268. physical. but nonlipid viruses and parvoviruses are less sensitive than lipid enveloped viruses (384). despite being widely studied. Although CRAs have been predominantly used as hard-surface disinfectants. but the exact mode of action is unknown. where the activity of CRAs is maximal. it is likely that iodine attacks the surface proteins of enveloped viruses. the most widely used are povidone-iodine and poloxamer-iodine in both antiseptics and disinfectants. whereas f2 capsid proteins could still adsorb to the host. 1999
ANTISEPTICS AND DISINFECTANTS
PHMB toward the negatively charged bacterial cell surface. This correlates with the observation that CRA activity is greatest when the percentage of undissolved HOCl is highest. In water. Less is known about the antiviral action of iodine. in solution. but they have been shown to inhibit oxygen uptake and induce leakage of amino acids (Table 2). By contrast. Their antibacterial properties and uses were reviewed by Hugo (213) and Hugo and Russell (226). novel acidiﬁed sodium chlorite (a two-component system of sodium chlorite and mandelic acid) has been described as an effective antiseptic (248). These problems were overcome by the development of iodophors (“iodine carriers” or “iodine-releasing agents”). 443). and chloramine-T. and PHMB is attracted to the inner membrane. (359) showed that chlorine inactivated naked f2 RNA at the same rate as RNA in intact phage. 485. Iodophors are complexes of iodine and a solubilizing agent or carrier. iodophors are considered less active against certain fungi and spores than are tinctures (454). CRAs also possess virucidal activity (34. 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. potentiation of oxidation may occur at low pH. 128. 116. 130). a number of studies have concluded that CRA-treated spores exhibit increased permeability of the spore coat (131. Although germicidal activity is maintained. Hypochlorous acid has long been considered the active moiety responsible for bacterial inactivation by CRAs.2-dibromo-4. sodium hypochlorite ionizes to produce Na and the hypochlorite ion. it was inferred that DNA synthesis was the sensitive target. In contrast. have been used as antibacterial agents. The exact mechanism of action of diamidines is unknown. 477). whereas above pH9. Floyd et al. Because concentrations below 5 mM (260 ppm) did not induce bacterial membrane disruption or extensive protein degradation. with chloramine-T being used to some extent. The isethionate salts of two compounds. but they may also destabilize membrane fatty acids by reacting with unsaturated carbon bonds (486). McKenna and Davies (321) described the inhibition of bacterial growth by hypochlorous acid. CRAs at higher concentrations are sporicidal (44.6 ppm). At 50 M (2. iodine is rapidly bactericidal. 407. diamidines are used for the topical treatment of wounds.4-diamidinodiphenoxypropane). The most important silver compound currently in use is silver sulfadiazine
. silver and its compounds have long been used as antimicrobial agents (55. and lysis occurs (268). 431). CRAs are highly active oxidizing agents and thereby destroy the cellular activity of proteins (42). 412). During treatment. the spore coat separates from the cortex. Similar to chlorine. 467. Although aqueous or alcoholic (tincture) solutions of iodine have been used for 150 years as antiseptics. Although less reactive than chlorine. OCl . the spores lose refractivity. tuberculocidal. Surprisingly. Chlorine-releasing agents. Iodine rapidly penetrates into microorganisms (76) and attacks key groups of proteins (in particular the freesulfur amino acids cysteine and methionine [184. and (iv) complete loss of membrane function follows. 421. as would be expected if they are considered as cationic surface-active agents. nucleotides. Diamidines The diamidines are characterized chemically as described in Table 1. Silver Compounds In one form or another. (iii) binding of PHMB to phospholipids occurs. with molecular iodine (I2) being primarily responsible for antimictrobial efﬁcacy (184). the active moiety. virucidal. Clinically. In addition.and iodine-based compounds are the most significant microbicidal halogens used in the clinic and have been traditionally used for both antiseptic and disinfectant purposes. Hypochlorous acid has also been found to disrupt oxidative phosphorylation (26) and other membrane-associated activity (70). 394. which culminates in cell death (184). at least seven iodine species are present in a complex equilibrium. the OCl ion having a minute effect compared to undissolved HOCl (130). with precipitation of intracellular constituents and a bactericidal effect. 12. Taylor and Butler (504) found that the RNA of poliovirus type 1 was degraded into fragments by chlorine but that poliovirus inactivation preceded any severe morphological changes. OCl predominates. chlorine exists predominantly as HClO. In addition. 184). HOCl completely inhibited the growth of E. This concept applies to hypochlorites. and DNA synthesis was inhibited by 96% but protein synthesis was inhibited by only 10 to 30%. 46. Damage to the cell surface of P. 412). Olivieri et al. even at low concentrations. 315. aqueous solutions are generally unstable. propamidine (4. In a particularly interesting paper.VOL. NaDCC. (149) and O’Brien and Newman (357) demonstrated that the capsid of poliovirus type 1 was broken down. The most important types of CRAs are sodium hypochlorite. the actual mechanism of action of CRAs is not fully known (Table 2). although increased penetration of outer cell layers may be achieved with CRAs in the unionized state.
PAA also decomposes to safe by-products (acetic acid and oxygen) but has the added advantages of being free from decomposition by peroxidases. its antiphage properties have been ascribed to the fact that AgSD binds to phage DNA (154. virucidal. Bacterial inhibition would then presumably be achieved when silver binds to sufﬁcient base pairs in the DNA helix. the precise mechanism of action of AgSD has yet to be solved. which causes irreversible cellular damage. including DNA (332. In addition to its effects on enzymes. 488) proposed that phenol acts only at the point of separation of pairs of daughter cells. being sporicidal. L-methionine. and of radioactivity from 14C-labeled E. The mechanism of the antimicrobial action of silver ions is closely related to their interaction with thiol (sulfydryl. silver acetate. Coagulation of cytoplasmic constituents at higher phenol concentrations. Pulvertaft and Lumb (386) demonstrated that low concentrations of phenols (0. Phenols Phenolic-type antimicrobial agents have long been used for their antiseptic. AgSD binds to cell components. Its main application is as a low-temperature liquid sterilant for medical devices. 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. Ag and sulfadiazine (SD). bacteria. yeasts. 509). 308). but it is also used as an environmental surface sterilant (100. 161. 164). REV. sodium bisulﬁte. although the signiﬁcance of this in terms of its lethal action is unclear (231. although other cellular components may be involved. the effects of hydrogen bond-breaking agents. coli (242. H2O2 is considered environmentally friendly. and outer cell layers all exhibit structural abnormalities. the microbial plasma or cytoplasmic membrane. with young bacterial cells being more sensitive than older cells to phenol. the presence of catalase or other peroxidases in these organisms can increase tolerance in the presence of lower concentrations. By contrast. 470). In recent years. and fungicidal at low concentrations ( 0. is an important target site for Ag activity (161. sterilization. 510. MICROBIOL. Ag inhibits cell division and damages the cell envelope and contents of P. 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. The Extra Pharmacopoeia (312). PAA probably denatures proteins and enzymes and increases cell wall permeability by disrupting sulfhydryl (™SH) and sulfur (S™S) bonds (22. amino acids containing disulﬁde (SS) bonds. although silver metal. cysteic acid. Phenol induces progressive leakage of intracellular constituents. Ag causes the release of K ions from microorganisms. because it can rapidly degrade into the innocuous products water and oxygen. 404). Hydrogen peroxide (H2O2) is a widely used biocide for disinfection. bactericidal. ﬂexible scopes. These and other ﬁndings imply that interaction of Ag with thiol groups in enzymes and proteins plays an essential role in bacterial inactivation. has been described by Hugo (215). aureus and E. Based on a chemical analysis. Similarly. Finally. coli. AgSD is essentially a combination of two antibacterial agents. greater activity is seen against gram-positive than gram-negative bacteria. Hugo and Bloomﬁeld (216. and the speciﬁcity of Ag for thiol groups were discussed in greater detail by Russell and Hugo (443) (Table 2). Peroxygens Hydrogen peroxide. Silver nitrate. Hydrogen bonding. unlike H2O2. It is a clear. Bacterial cells increase in size. including the release of K . are listed in Martindale. unlike silver nitrate. It has been known for many years (215) that. 392. Chlorocresol has a similar action (124). depending on the compound. although they have often been referred to as “general protoplasmic poisons. 49. In general. 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). and sulfur-containing compounds such as cystathione. produces surface and membrane blebs in susceptible (but not resistant) bacteria (96). aeruginosa (398). H2O2 demonstrates broad-spectrum efﬁcacy against viruses. H2O2 acts as an oxidant by producing hydroxyl free radicals (•OH) which attack essential cell components. Silver sulfadiazine. and antisepsis. 308). the Ag ion interacts with nucleic acids (543). and DNA. although this activity is signiﬁcantly increased in the gaseous phase. although other target sites remain a possibility (397. AgSD has a broad spectrum of activity and. It has been proposed that exposed sulfhydryl groups and double bonds are particularly targeted (38). most contain stabilizers to prevent decomposition. 38). and silver protein. non-sulfurcontaining amino acids. aeruginosa.032%.
(AgSD). although without any blebs (protuberances) (398). colorless liquid that is commercially available in a va-
riety of concentrations ranging from 3 to 90%. disinfectant. 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. ™SH) groups (32. Lukens (292) proposed that silver salts and other heavy metals such as copper act by binding to key functional groups of fungal enzymes. however. Ag produces other changes in microorganisms. Similar to H2O2. 265). Peracetic acid. and sodium thiosulfate were all unable to neutralize Ag activity. it interacts preferentially with the bases in DNA rather than with the phosphate groups. Silver nitrate causes marked inhibition of growth of Cryptococcus neoformans and is deposited in the vacuole and cell wall as granules (60). 320 g/ml) and other (nonphenolic) agents lysed rapidly growing cultures of E. 547). Srivastava and Thompson (487. or preservative properties. 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). cytoplasmic contents.” they have membrane-active properties which also contribute to their overall activity (120) (Table 2). and streptococci and concluded that autolytic enzymes were not involved. taurine. staphylococci. with which is associated many important enzymes. 329. and hemodialyzers. including lipids. Virucidal properties might also be explained by binding to ™SH groups (510). Their antifungal action probably involves damage to the plas-
. proteins. thereby inhibiting transcription. Although pure solutions are generally stable. and remaining active in the presence of organic loads (283. The phenolics possess antifungal and antiviral properties. and the cytoplasmic membrane. Fentichlor affected the metabolic activities of S. Higher concentrations of H2O2 (10 to 30%) and longer contact times are required for sporicidal activity (416). silver nitrate.156
MCDONNELL AND RUSSELL
CLIN. Peracetic acid (PAA) (CH3COOOH) is considered a more potent biocide than hydrogen peroxide. silver compounds have been used to prevent the infection of burns and some eye infections and to destroy warts. all of which have antimicrobial properties.3%) (38). 388). and bacterial spores (38). Clearly. 387. the ﬁrst index of membrane damage (273).
the cationic agents. the MBCs were not affected. aeruginosa and molds and are sporostatic toward bacterial spores. 319). aeruginosa PAO by bacteriophage F116 (301).5-dimethylphenol. water-repellent (hydrophobic) group and the other a water-attracting (hydrophilic or polar) group. It induces leakage. There is thus a loss of structural organization and integrity of the cytoplasmic membrane in bacteria. has no effect on phage DNA within the capsid. nonionic. a prominent 14:1 fatty acid was absent in the resistant strain. have meant that its use in antiseptic products has been limited. as for other phenols. Studies with a divalent-ion-dependent E. The cationic agents react with phospholipid components in the cytoplasmic membrane (69). they exhibit broad-spectrum efﬁcacy but have little activity against P. Triclosan. is to inhibit the membranebound part of the electron transport chain. The primary action of hexachlorophene.2 -dihydroxy-3. especially in antiseptic soaps and hand rinses. which drives these ancillary activities (428). but P. Phenol does not affect the transduction of P.4 -trichloro-2 -hydroxydiphenyl ether. while higher. and inhibits respiration. Depending on the basis of the charge or absence of ionization of the hydrophilic group. The bacterial cytoplasmic membrane provides the mechanism whereby metabolism is linked to solute transport. thereby producing membrane distortion and protoplast lysis under osmotic stress. 241.6 -hexachlorodiphenylmethane) is another bis-phenol whose mode of action has been extensively studied. Hexachlorophene (hexachlorophane. 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). (ii) reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization.5. ﬂagellar movement. and (v) wall lysis caused by autolytic enzymes. Both compounds have been shown to have cumulative and persistent effects on the skin (313). It was proposed that divalent ions and fatty acids may adsorb and limit the permeability of triclosan to its site of action (370). Because of its phenolic nature. and there were minor differences in other fatty acid species.4. The combined potential (concentration or osmotic effect of the proton and its electropositivity) is the PMF. 304).e. (iv) degradation of proteins and nucleic acids.VOL. membrane damage. resulting in leakage of intracellular constituents. application to mucous membranes. 481). anionic. coli and S. 521). 432). Furthermore. 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 excellent for hard-surface cleaning and deodorization. The threshold concentration for the bactericidal activity of hexachlorphene is 10 g/ml (dry weight). Chloroxylenol is bactericidal. based on studies with Bacillus megatherium. rather than speciﬁc.. Quaternary Ammonium Compounds Surface-active agents (surfactants) have two regions in their molecular structures. it would be expected to have an effect on microbial membranes.6. 215. QACs cause lysis of spheroplasts and protoplasts suspended in sucrose (107. In general. Hexachlorophene. together with other damaging effects to the bacterial cell (120). but it has been suggested that the primary effects are on the cytoplasmic membrane.. 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. The QAC cetrimide was found (121) to have an effect on the PMF in S. in particular in neonates. and the generation of ATP. whereas trichlorosalicylanide induces lysis in ammonium nitrate by increasing NO3 permeability (428). In contrast. however. 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. QACs have been used for a variety of clinical purposes (e.3 . suggesting. The speciﬁc mode of action of triclosan is unknown. Irgasan DP 300) exhibits particular activity against grampositive bacteria (469. concerns about toxicity (256). Triclosan and hexachlorophane are the most widely used biocides in this group. Bis-Phenols The bis-phenols are hydroxy-halogenated derivatives of two phenolic groups connected by various bridges (191. (iii) leakage of intracellular low-molecular-weight material. and the other effects noted above are secondary ones that occur only at high concentrations (92.5 . 12. In studies with E. Halophenols Chloroxylenol (4-chloro-3. Of these. bactericidal concentrations resulted in the rapid release of cellular components and cell death (393). At its bacteriostatic concentration. causes protoplast lysis. Despite the broad-spectrum efﬁcacy of hexachlorophene. because the membrane distortion is not sufﬁciently drastic. 243. hexachlorophene is bactericidal at 0°C despite causing little leakage at this temperature. The non-QAC agents TCC and trichlorosalicylanide have speciﬁc effects: TCC induces protoplast lysis in ammonium chloride by increasing Cl permeability. Isolated membranes do not undergo disaggregation on exposure to QACs. one a hydrocarbon. Speciﬁcally. Protons are extruded to the exterior of the bacterial cell during metabolism. p-chloro-m-xylenol) is the key halophenol used in antiseptic or disinfectant
formulations (66). Surprisingly. but peak leakage occurs at concentrations higher than 50 g/ml and cytological changes occur above 30 g/ml. 446). It has been known for many years that QACs are membraneactive agents (221) (Table 2) (i. preoperative disinfection of unbroken skin. 1999
ANTISEPTICS AND DISINFECTANTS
ma membrane (436). aureus strains for which the triclosan MICs were elevated. with a target site predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane in yeasts) (215). 158.g. and ampholytic (amphoteric) compounds. as exempliﬁed by quaternary ammonium compounds (QACs). Triclosan (2. Minor changes in fatty acid proﬁles were recently found in both E. its mechanism of action has been little studied despite its widespread use over many years. They are sometimes known as cationic detergents. 428). 522). are the most useful antiseptics and disinfectants (160).
. QACs (and chlorhexidine) induce lysis of protoplasts or spheroplasts suspended in various solutes because they effect generalized. triclosan in combination with EDTA caused increased permeability of the outer membrane (282). Its efﬁcacy against gram-negative bacteria and yeasts can be signiﬁcantly enhanced by formulation effects. and has little effect on several of the phage band proteins unless treatments of 20 min or longer are used (303. triclosan at subinhibitory concentrations inhibited the uptake of essential nutrients. that the cumulative effects on multiple targets contribute to the bactericidal activity (318. and disinfection of noncritical surfaces). aureus. surfactants are classiﬁed into cationic. For example. 2. coli. aeruginosa and many molds are highly resistant (66. triclosan may have anti-inﬂammatory activity (25. In addition to having antimicrobial properties. Reports have also suggested that in addition to its antibacterial properties.
The asterisk indicates that the conclusions are not yet universally agreed upon. The initial toxic effect of QACs on yeast cells is a disorganization of the plasma membranes. and bacterial spores) to antibacterial agents (43. Vapor-phase hydrogen peroxide and PAA are considered more active (as oxidants) at lower concentrations than in the liquid form (334). and activity at lower temperature. and toxic residuals from the sterilization procedure can be routinely eliminated by correct aeration. different types of microorganisms vary in their response to antiseptics and disinfectants. protozoa. transmissible DNA cassettes). constitutively synthesized enzymes may bring about degradation of a compound (43. Both active agents are used in combination with gas plasma in low-temperature sterilization systems (314). As a result. Intrinsic Bacterial Resistance Mechanisms For an antiseptic or disinfectant molecule to reach its target site. The nature and composition of these layers depend on the organism type and may act as a permeability barrier. As alkylating agents. and prions separately. Acquired. The most widely used active agents in these “cold” systems are ethylene oxide. however. 307). and hydrogen peroxide) or by vaporphase sterilization systems (Table 1). Bacterial Resistance to Antiseptics and Disinfectants In recent years. their activity is dependent on active concentration. rapid action. viruses. acquired resistance to certain other types of biocides has been observed. 1. Formaldehyde gas is similar and has the added advantage of being nonexplosive but is not widely used in health care. the disadvantages include limited penetrability and applications. considerable progress has been made in understanding more fully the responses of different types of bacteria (mycobacteria. This aspect of QACs is considered below (see “Intrinsic resistance of gram-negative bacteria”). PAA. 84. Intrinsic (innate) resistance
. REV. In studies with different phages (298–301. Furthermore. and other organic compounds. with recent work. CPC signiﬁcantly inhibited transduction by bacteriophage F116 and inactivated the phage particles. Their main advantages over other vapor-phase systems include low toxicity. although the actual effects on mycobacteria have been little studied (419). duration of exposure. formaldehyde and. nonsporulating bacteria. 214. QAC-based products induced disintegration and morphological changes of human HBV. In recent years. The QACs have an effect on lipid. both are particularly reactive with sulfhydryl and other enzyme-reactive groups. Because different types of organisms react differently. fungi. 1). 419. The QAC cetylpyridium chloride (CPC) induces the leakage of K and pentose material from the yeast S. 486). with organized lipid structures in the membranes (and in lipid bilayers) being disrupted. QACs are sporostatic. Intrinsic resistance is demonstrated by gramnegative bacteria. 422. the outer layers of a cell must be crossed.
differences. they attack proteins. staphylococci (Table 5). 485. the QACs are not mycobactericidal but have a mycobacteriostatic action. nucleic acids. CPC altered the protein bands of F116 but did not affect the phage DNA within the capsid. Alternatively but less commonly.
QACs are also believed to damage the outer membrane of gram-negative bacteria. composition. MECHANISMS OF RESISTANCE Introduction As stated above. Ethylene oxide gas has the disadvantages of being mutagenic and explosive but is not generally harsh on sensitive equipment. However. no biphasic effect on protoplast lysis was observed. temperature. and relative humidity (87). this classiﬁcation can be further extended (Fig. Unlike chlorhexidine. in which there may be a reduced uptake (422. 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). hydrogen peroxide and PAA. MICROBIOL. and physiology. albeit by an unknown mechanism (414).158
MCDONNELL AND RUSSELL
CLIN. 415. under certain conditions. and. resulting in loss of infectivity (382). extrachromosomal DNA) or transposons (chromosomal or plasmid integrating. 358). enveloped (including human immunodeﬁciency virus and HBV) but not nonenveloped viruses (394. 428). Descending order of resistance to antiseptics and disinfectants. microbial susceptibility to antiseptics and disinfectants has been classiﬁed based on these
FIG. more recently developed. thereby promoting their own uptake. Traditionally. resistance can be either a natural property of an organism (intrinsic) or acquired by mutation or acquisition of plasmids (self-replicating. notably in staphylococci. bacterial spores. plasmidmediated resistance is most widely associated with mercury compounds and other metallic salts. it is convenient to consider bacteria. 496). mycobacteria. This is hardly surprising in view of their different cellular structure. Likewise. cerevisiae and induces protoplast lysis as well as interacting with crude cell sap (205). 303–305. 414. Vapor-Phase Sterilants Many heat-sensitive medical devices and surgical supplies can be effectively sterilized by liquid sterilants (in particular glutaraldehyde. Ethylene oxide and formaldehyde are both broadspectrum alkylating agents.
the vegetative cell (stage 0) undergoes a series of morphological changes that culminate in the release of a mature spore (stage VII). phenolics. their spores are widely used as indicators of efﬁcient sterilization. The cortex consists largely of peptidoglycan. 420. DNA. and phenylmercuric nitrate. are present in the spore protoplast. 12. 474). Sporulation itself is a process in which a vegetative cell develops into a spore and involves seven stages (designated 0 to VII). The association of the onset of resistance to a particular antiseptic or disinfectant with a particular stage(s) in spore development is thereby demonstrated. sodium lauryl sulfate. 2). The spore coats comprise a major portion of the spore. 375. the order of development of resistance was toluene (marker). but high concentrations may be necessary to achieve a sporicidal effect (e. chlorhexidine gluconate. potassium. even high concentrations of alcohol. 150. diamines Chlorhexidine. phenol. and glutaraldehyde. C. Several techniques are available for studying mechanisms of spore resistance (428). aureus
E. In brief. QACs Glutaraldehyde Chlorhexidine. Although Bacillus species are generally not pathogenic. glycocalyx may also be involved Waxy cell wall prevents adequate biocide entry Reason for high resistance of some strains of M. QACs.. including a spore-speciﬁc muramic lactam.000 100 2 1 0. Resistance to antiseptics and disinfectants develops during sporulation and may be an early. A thin exosporium may be present in the spores of some species but may surround just one spore coat. 378. outside which are the inner and outer spore coats. Intrinsic resistance mechanisms in bacteria to antiseptics and disinfectants
Type of resistance
Mechanism of resistance
Impermeability Gram-negative bacteria Mycobacteria Bacterial spores Gram-positive bacteria
QACs.5 2. including the vegetative cells of Bacillus and Clostridium species. aeruginosa
Benzalkonium chloride Benzethonium chloride Cetrimide Chlorhexidine Hexachlorophene Phenol o-Phenylphenol Propamine isethionate Dibromopropamidine isethionate Triclosan
0. From these studies (Fig. and DPA. adding an antiseptic or disinfectant at the commencement of
sporulation and determining how far the process can proceed. which are rapidly degraded during germination. produced by treatment of spores un-
TABLE 6. These structures consist largely of protein. are all relevant to the mechanism(s) of resistance presented by bacterial spores to antiseptics and disinfectants. 418. or (very) late event (103. the germ cell (protoplast or core) and germ cell wall are surrounded by the cortex. These aspects. triclosan. Also present are large amounts of low-molecular-weight basic proteins (small acid-soluble spore proteins [SASPs]).000 500 64 4 5
250 250 64–128 5–60 250 2. During this process. 422.5 0. with an alkali-soluble fraction made up of acidic polypeptides being found in the inner coat and an alkali-resistant fraction associated with the presence of disulﬁde-rich bonds being found in the outer coat. Useful markers for monitoring the development of resistance are toluene (resistance to which is an early event). 414.1
50 32 16 1 12. Many biocides are bactericidal or bacteristatic at low concentrations for nonsporulating bacteria. and phosphorus. heat (intermediate).5–1 0. such as staphylococci (Table 6). 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. intermediate. coli
P. chromosomally controlled property of a bacterial cell that enables it to circumvent the action of an antiseptic or disinfectant. lysozyme (marker).g. and chlorhexidine lack a sporicidal effect. difﬁcile is the most common cause of hospitalacquired diarrhea (478). A typical spore has a complex structure (29. 1999
ANTISEPTICS AND DISINFECTANTS TABLE 5.000 256 32 300
10). sodium hypochlorite. for example. and lysozyme (late) (236. phenolics Chlorhexidine
Barrier presented by outer membrane may prevent uptake of antiseptic or disinfectant. and mercuric chloride. 474). 237).000 1. Clostridium species are signiﬁcant pathogens.5 4 0. 423.VOL. and its Spo mutants have helped determine the stages at which resistance develops (262. m-cresol. subtilis strain. and examining the role of SASPs. chlorocresol. 457). By contrast. formaldehyde. although this may be achieved when these compounds are used at elevated temperatures (475). 46. Studies with a wild-type B. 151). manganese. Gram-negative bacteria tend to be more resistant than gram-positive organisms. 429. especially the roles of the coat(s) and cortex. QACs. employing spore mutants that do not sporulate beyond genetically determined stages in sporulation. They include removing the spore coat and cortex by using a “step-down” technique to achieve a highly synchronous sporulation (so that cellular changes can be accurately monitored). 168. cetylpyridinium chloride. 375. MIC of some antiseptics and disinfectants against gram-positive and gram-negative bacteriaa
Chemical agent MIC ( g/ml) for: S. Such procedures have helped provide a considerable amount of useful information.
Based on references 226 and 440. sodium dichloroisocyanurate. MICs of cationic agents for some MRSA strains may be higher (see Table
. chelonae(?) Spore coat(s) and cortex present a barrier to entry of antiseptics and disinfectants Glycocalyx/mucoexopolysaccaride may be associated with reduced diffusion of antiseptic Breakdown of chlorhexidine molecule may be responsible for resistance
Inactivation (chromosomally mediated)
is thus a natural. as well as most of the calcium. for glutaraldehyde and CRAs). Stages IV (cortex development) to VII are the most important in the development of resistance to biocides.5 2. Spore coat-less forms. and moist heat (marker). Intrinsic resistance of bacterial spores. RNA.
SASPs contribute to spore resistance to peroxide and hypochlorite but may not be the only factors involved. contain an aldehyde group(s) and are alkylating agents. plasmid. since cells must reach stages V to VI (synthesis of spore coats and maturation) to attain heat resistance but only stage III (forespore engulfment) to attain toluene resistance (Fig. Since glutaraldehyde and the monoaldehyde.
der alkaline conditions with urea plus dithiothreitol plus sodium lauryl sulfate (UDS). 530). By contrast. subtilis. 419). 484) demonstrated that formaldehyde-exposed spores of B. Experiments designed to distinguish between germination and outgrowth in the revival process have demonstrated that sodium hydroxide-induced revival increases the potential for germination. even though the dialdehyde is a more powerful alkylating agent. If this were true. The initial development and maturity of the cortex are implicated in the development of resistance to phenolics. NaDCC. Although neither aspect is truly a resistance mechanism. Based on other ﬁndings. 380). chlorine. Also present are complex lipids. They found that chlorhexidine affected spore development. 390. it could also be assumed that
spores would exhibit the same resistance mechanisms for these disinfectants. A small proportion of glutaraldehyde-treated spores of various Bacillus species were revived when the spores were treated with alkali after neutralization of glutaraldehyde with glycine (103. 461. on the development of spores of B. 322. 524). Spicher and Peters (483. 106. subtilis 168 MB2 were investigated by Knott and Russell (261). as concentrations of the biguanide increased. Thus. and iodine was examined by Russell and his colleagues (103. The revival of disinfectant-treated spores has not been extensively studied. Pathogenic bacteria such as Mycobacterium tuberculosis exist in a relatively nutrient-rich environment. formaldehyde forms a glycol in equilibrium (512. Alkaline glutaraldehyde does not readily form glycols in aqueous solution (178). thereby protecting it from attack by enzymes and antimicrobial agents.
. These ﬁndings demonstrate that the spore coats have an undoubted role in conferring resistance but that the cortex also is an important barrier since (UDS plus lysozyme)-treated spores are much more sensitive to chlorineand iodine-releasing agents than are UDS-exposed spores. indicative of high levels of mature spores. 105. stearothermophilus casts further doubt on the efﬁcacy of low-temperature steam with formaldehyde as a sterilizing procedure (541). The peptidoglycan is covalently linked to the polysaccharide copolymer (arabinogalactan) made up of arabinose and galactose esteriﬁed to mycolic acids. sodium dichloroisocyanurate. since the coats and cortex also play a role (428). 1) (177. CPC. subtilis could be revived after a subsequent heat shock process. Two other aspects of spores should be considered: the revival of injured spores and the effects of antiseptics and disinfectants on germinating and outgrowing spores. Development of resistance of Bacillus subtilis during sporulation.
FIG. A more recent ﬁnding with B. 424. 2. and resistance to glutaraldehyde may be linked to coat formation (262). The revival of spores exposed to glutaraldehyde. Setlow and his coworkers (472) demonstrated that / -type SASPs coat the DNA in wild-type spores of B. Spores ( ) lacking these / -type SASPs are signiﬁcantly more sensitive to hydrogen peroxide (471) and hypochlorite (456). chlorhexidine. Mycobacteria are well known to possess a resistance to antiseptics and disinfectants that is roughly intermediate between those of other nonsporulating bacteria and bacterial spores (Fig. glutaraldehyde. Roman numerals indicate the sporulation stage from III (engulfment of the forespore) to VII (release of the mature spore). 2). cetylpyridinium chloride. Likewise. each can provide useful information about the site and mechanism of action of sporicidal agents and about the associated spore resistance mechanisms and might be of clinical importance. formaldehyde could well be acting poorly as an alcohol-type disinfectant rather than as an aldehyde (327). 377. 376. formaldehyde. 532–537). 356). The slightly increased resistance to toluene compared to resistance to heat was not surprising. the germination process is also implicated in the revival of spores exposed to other disinfectants. if sporulation is inhibited by chlorhexidine. In aqueous solution. spore index values (the percentage of cells forming spores) decreased and sensitivity to both heat and toluene increased. While less deﬁnitive than the earlier approaches. these procedures provide further evidence of the involvement of the cortex and coats in chlorhexidine resistance. Bloomﬁeld and Arthur (44. lipopolysaccharides (LPSs). MICROBIOL. The cell wall composition of a particular species may be inﬂuenced by its environmental niche (27). The mycobacterial cell wall is a highly hydrophobic structure with a mycoylarabinogalactan-peptidoglycan skeleton (27. this resistance is enhanced in developing spores by the initiation of spore coat synthesis (262). The effect of various concentrations of chlorhexidine. 345. This appears to be at odds with the extremely late development of resistance to the dialdehyde. Resistance to formaldehyde may be linked to cortex maturation. sublethal to vegetative bacteria. it would be plausible to assume that they would have a similar mode of sporicidal action. Intrinsic resistance of mycobacteria. To date. There is no evidence that enzymatic degradation of harmful molecules takes place. However. REV. Arabic numbers indicate the time (hours) following the onset of sporulation and the approximate times at which resistance develops against biocides (262). formaldehyde. 389. whereas saprophytic mycobacteria living in soil or water are exposed to natural antibiotics and tend to be more intrinsically resistant to these drugs. including porin channels through which hydrophilic molecules can diffuse into the cell (232. 45) and Bloomﬁeld (43) showed that this treatment also removes a certain amount of cortex and that the amount of cortex remaining can be further reduced by the subsequent use of lysozyme. more cells are likely to reach stage III than the later stages. thus. 379. Development of resistance during sporulation to formaldehyde was an early event but depended to some extent on the concentration (1 to 5% [vol/vol]) of formaldehyde used. the control (untreated) culture was highly resistant to both of these agents and had a high spore index value. Similar cell wall structures exist in all the mycobacterial species examined to date (228). in other words.160
MCDONNELL AND RUSSELL
CLIN.or transposon-mediated resistance to biocides has not been demonstrated in mycobacteria. and proteins. CHG. it is now clear that cortex development is at least partially responsible for resistance to chlorhexidine and QACs. The most likely mechanism for the high resistance of mycobacteria is associated with their complex cell walls that provide an effective barrier to the entry of these agents. have also been of value in estimating the role of the coats in limiting the access of antiseptics and disinfectants to their target sites.
chelonae. 319). avium and M. the activity of chlorhexidine and of a QAC. these membrane-active agents. and chlorhexidine. M. it was proposed (T. Other workers have also observed an above-average resistance of M. There is no evidence to date that vancomycin-resistant enterococci or enterococci with high-level resistance to aminoglycoside antibiotics are more resistant to disinfectants than are antibiotic-sensitive enterococcal strains (9. 455). One species of mycobacteria currently causing concern is M. cited in reference 99) that the resistance of mycobacteria to QACs was related to the lipid content of the cell wall. nonmycobacterial grampositive bacteria (Fig. Intrinsic resistance of other gram-positive bacteria. It is not possible. These ﬁndings support the concept of the cell wall acting as a permeability barrier to these drugs (425). lysozyme-induced protoplasts of these cells remained sensitive to. the plasticity of the bacterial cell envelope is a well-known phenomenon (381). 451). From these data. a newer formulation (Sactimed-I-Sinald) containing a mixture of alkyl polyguanides and alkyl QACs is claimed to be mycobactericidal (211. in contrast. there is some doubt whether the antibacterial agents had been properly quenched or neutralized to prevent carryover of inhibitory concentrations into recovery media. Methyl-4-(2-octadecylcyclopropen-1-yl) butanoate (MOCB) is a structural analogue of a key precursor in mycolic acid synthesis. benzethonium. There is no evidence to date that uptake of glutaraldehyde by M. It was also noted that the resistance of various species of mycobacteria was related to the content of waxy material in the wall. to comment on other components. 440. chelonae to glutaraldehyde and formaldehyde (72) but not to PAA (187. However. the effects of MOCB on mycolic acid synthesis and m-FL-phe and ethambutol on outer wall biosynthetic processes leading to changes in cell wall architecture appear to be responsible for increasing the intracellular concentration of chemotherapeutic drugs. 422. By contrast. Therefore. 2) (428. 52. either as a physical barrier to disinfectant penetration or as a loose layer interacting with or absorbing the biocide molecules. chelonae is known to adhere strongly to smooth surfaces. 455). an inhibitor of arabinogalactan (391. In nature. aureus. Since highmolecular-weight substances can readily traverse the cell wall of staphylococci and vegetative Bacillus spp. However. the activity of these can be substantially increased by formulation effects. inhibition of myocide C. Many years ago. Neither of these appears to act as an effective barrier to the entry of antiseptics and disinfectants. 425. aeruginosa is considerably more resistant to most of these agents. cetrimide. and were lysed by. although some information is available. However. The cell wall of staphylococci is composed essentially of peptidoglycan and teichoic acid. aureus and E.. However. hydrogen peroxide. 417. 440). Under such circumstances. For example. The reasons for this high glutaraldehyde resistance are unknown. such as chlorhexidine and QACs. Gram-negative bacteria are generally more resistant to antiseptics and disinfectants than are nonsporulating. P. including chlorhexidine. which has a low total cell lipid content. the cell wall in whole cells is responsible for their modiﬁed response. diamidines. and triclosan but little difference in chlorhexidine susceptibility. tuberculosis can be potentiated in the presence of ethambutol (52). tuberculosis. For example. Ethambutol. 501) and phospholipid (461. The outer membrane of gram-negative bacteria acts as a barrier that limits the entry of many chemically unrelated types
. 294). 52). removal of slime by washing rendered the cells sensitive. low concentrations of antiseptics and disinfectants such as chlorhexidine must presumably traverse this permeability barrier. also disorganizes these layers. a number of QAC-based products claim to have mycobacterial activity. enterococci are generally less sensitive to biocides than are staphylococci. In addition. the slime plays a protective role. 462) synthesis. which may render cells within a bioﬁlm less susceptible to disinfectants. Thus. which has a higher lipid content. since these organisms are sometimes isolated from endoscope washes and dialysis water. 17. 353). 12. 1999
ANTISEPTICS AND DISINFECTANTS
Antiseptics or disinfectants that exhibit mycobacterial activity are phenol. avium-intracellulare may be particularly resistant (51. 441). and differences in inhibitory and bactericidal concentrations have also been found among enterococcal species (257). chelonae strain demonstrated an increased tolerance to PAA but not to NaDCC or to a phenolic. Mycobacterium phlei. since these have yet to be investigated. there is a marked difference in the sensitivity of S. coli to QACs (benzalkonium. 48. and mycolic acid biosynthesis enhances drug susceptibility. cetylpyridinium chloride. Gilbert and Brown (171) demonstrated that the sensitivity of Bacillus megaterium cells to chlorhexidine and 2-phenoxyethanol is altered when changes in growth rate and nutrient limitation are made with chemostat-grown cells. 11. other wellknown bactericidal agents. However. Therefore. 428. However. avium to drugs (391). However. a reference strain showed a 5-log-unit reduction after a contact time of 10 min (519). S. possess an above-average resistance to cationic agents such as chlorhexidine and QACs (311. Shen. Growth rate and any growthlimiting nutrient will affect the physiological state of the cells. It is now known that because of the highly hydrophobic nature of the cell wall. H. arabinogalactan. 419. with the cells surrounded by a slime layer. alcohol. 425. produces signiﬁcant alterations in the outer cell wall layers (106). and glutaraldehyde (16. aureus may exist as mucoid strains. However. It would be useful to have information about the uptake into the cells of these antiseptic agents in the presence and absence of different cell wall synthesis inhibitors. this may explain the sensitivity of these organisms to many antibacterial agents including QACs and chlorhexidine (411. ethambutol induces the formation of ghosts without the dissolution of peptidoglycan (391). Fewer studies have been made of the mechanisms involved in the resistance of mycobacteria to antiseptics and disinfectants. although M. hydrophilic biocides are generally unable to penetrate the mycobacterial cell wall in sufﬁciently high concentrations to produce a lethal effect. 99. at present. Nonmucoid strains are killed more rapidly than mucoid strains by chloroxylenol. because the MICs are of the same order as those concentrations inhibiting the growth of nonmycobacterial strains such as S. Thus. Treatment of this organism with m-ﬂuoro-DL-phenylalanine (m-FL-phe). In support of this contention.VOL. which inhibits mycocide C synthesis. chelonae is reduced. One such strain was not
killed even after a 60-min exposure to alkaline glutaraldehyde. The component(s) of the mycobacterial cell wall responsible for the high biocide resistance are currently unknown. and (not shown) Proteus spp. This glutaraldehyde-resistant M. but there is little difference in killing by phenols or chlorinated phenols (263). Intrinsic resistance of gram-negative bacteria. and cetrimide). was more sensitive than M. against M. Inhibitors of cell wall synthesis increase the susceptibility of M. Examples of MICs against gram-positive and -negative organisms are provided in Table 6. 419. Based on these data. the thickness and degree of crosslinking of peptidoglycan are likely to be modiﬁed and hence the cellular sensitivity to antiseptics and disinfectants will be altered. are mycobacteristatic even when used at high concentrations (51. hexachlorophene. PAA. it may be inferred that arabinogalactan is one cell wall component that acts as a permeability barrier to chlorhexidine and QACs.
276. because of their small size. also. 355. Proteus spp. and high-level resistance to chlorhexidine formed the basis of a series of studies of the resistance mechanism(s) (86. thereby promoting their own uptake (197). coli. but hydrophobic molecules diffuse across the outer membrane bilayer (Table 7). 73. the high content of phosphate-linked arabinose in its LPS decreases the afﬁnity of the outer membrane for polymyxin antibiotics and other cationic and polycationic molecules (97. 516. 440). the porins may not permit general diffusion through them. In addition to these hydrophilic and hydrophobic entry pathways.. inner membrane. (230) could ﬁnd no such role with chlorhexidine-resistant P. and Providencia stuartii (428. 135. 516). P. aeruginosa is responsible for its high resistance. and diamidines. deﬁned as a consortium of organisms organized within an extensive exopolysaccharide exopolymer (93. 381). and 439. P. Possible transport of some antiseptics and disinfectants into gram-negative bacteriaa
CLIN. furthermore. damage to IM enables biocide to enter cytosol. Smooth. Strains of P. 517). 600) hydrophilic molecules readily pass via the porins into gram-negative cells. but high phenolic concentrations coagulate cytoplasmic constituents
Data from references 197. which implies that such agents have little difﬁculty in crossing the outer layers of the cells of this organism. by contrast.
Passage across OMb
Passage across IMb
Chlorhexidine QACs Phenolics
Self-promoted uptake(?) Self-promoted uptake(?). 433–435. and Achromobacter/Alcaligenes xylosoxidans (358). intact LPS molecules prevent ready access of hydrophobic molecules to phospholipid and thence to the cell interior.
of antibacterial agents (18. It is conceivable that the stretched nature of both the outer and inner membranes in -lactam-treated organisms could contribute to this increased susceptibility. 178. biguanidies. however. and diamidines have been isolated from clinical sources.
. there is little evidence to implicate the inner membrane in biocide resistance. and it was concluded that (i) subtle changes in the structural arrangement of the cell envelopes of these strains was associated with this resistance and (ii) the inner membrane was not implicated (230). Low-molecular-weight (Mr ca. which are inherently more sensitive to many antiseptics and disinfectants. EDTA. Bioﬁlms can consist of monocultures. Gram-negative bacteria that show a high level of resistance to many antiseptics and disinfectants include P. QACs. It must be added. Lannigan and Bryan (275) proposed that decreased susceptibility of Serratia marcescens to chlorhexidine was linked to the inner membrane.162
MCDONNELL AND RUSSELL TABLE 7. typhimurium mutants are more sensitive to chlorhexidine than are wild-type strains (433). B. 197. that the QACs and diamidines are considerably less active against wildtype strains than against deep rough strains whereas chlorhexidine has the same order of activity (MIC increase about 2 to 3 fold) against both types of E. stuartii. outer membrane. At present. 440. in comparison with other organisms. OM. Physiological (phenotypic) adaption as an intrinsic mechanism. Bioﬁlms are important for several reasons. or of mixed phenotypes of a given species (57. Few authors have considered peptidoglycan in gram-negative bacteria as being a potential barrier to the entry of inhibitory substances. 411) that penicillin-induced spheroplasts and lysozyme-EDTA-Tris “protoplasts” of gram-negative bacteria are rapidly lysed by membrane-active agents such as chlorhexidine. Some excellent publications that deal with the nature. stuartii that showed low-. It is claimed that these damage the outer membrane. In addition. The outer membrane of P. This membrane is composed of lipoprotein and would be expected to prevent passive diffusion of hydrophilic molecules. coli (520). 496). 438). Some strains that are highly resistant to chlorhexidine. coli strains (434. by contrast. Pseudomonas stutzeri. the phospholipid patches at the cell surface have their head groups oriented toward the exterior. there have been instances (discussed in reference 422) where gram-negative organisms grown in subinhibitory concentrations of a penicillin have deﬁcient permeability barriers. 435. 196. of several diverse species. Like Proteus spp.. S. 433 to 435. because of the phospholipid patches on the cell surface. 409. The peptidoglycan content of these organisms is much lower than in staphylococci. MICROBIOL. aeruginosa. and P. A particularly troublesome member of the genus Providencia is P. 538). Furthermore. In wild-type gram-negative bacteria. but Ismaeel et al. and content of bioﬁlms are available (125. S. The presence of a less acidic type of
outer membrane LPS could be a contributing factor to this intrinsic resistance (97. These mutants tend to be hypersensitive to hydrophobic antibiotics and disinfectants. typhimurium. Nevertheless. where further interaction occurs IM is a major target site. which lack the O-speciﬁc side chain and most of the core polysaccharide. 422. 439). 428). The high Mg2 content aids in producing strong LPS-LPS links. stuartii strains have been isolated from urinary tract infections in paraplegic patients and are resistant to different types of antiseptics and disinfectants including chlorhexidine and QACs (492. 366. cepacia is often considerably more resistant in the hospital environment than in artiﬁcial culture media (360). It is also known that changes in membrane composition affect sensitivity to ethanol (159). it has been known for many years (215. where further interaction occurs IM is a major target site. REV. deep rough (heptose-less) mutants are hydrophobic. Burkholderia cepacia. 169. 438. However. is highly sensitive to many antibiotics and disinfectants (449). Gross differences in the composition of the outer layers of these strains were not detected. 94). a third pathway has been proposed for cationic agents such as QACs. formation. The association of microorganisms with solid surfaces leads to the generation of a bioﬁlm. The possibility exists that the cytoplasmic (inner) membrane provides one mechanism of intrinsic resistance. wild-type bacteria have a hydrophobic cell surface. aeruginosa. IM. Members of the genus Proteus are invariably insensitive to chlorhexidine (311). 516). OM might present a barrier Hydrophobic pathway (activity increases as hydrophobicity of phenolic increases)
IM is a major target site. intermediate-. chlorhexidine degradation was reported for S. stuartii. marcescens. Polycations disorganize the outer membrane of E. In deep rough strains. damage to IM enables biocide to enter cytosol. aeruginosa (134. This conclusion is based on the relative sensitivities of staphylococci and gram-negative bacteria and also on studies with outer membrane mutants of E. there are differences in LPS composition and in the cation content of the outer membrane (54).
. and foci for contamination of hygienic products (10.... cepacia in chlorhexidine and by Anderson et al. Bioﬁlms and microbial response to antimicrobial agents
Mechanism of resistance associated with bioﬁlms
Exclusion or reduced access of antiseptic or disinfectant to underlying cell... a novel strategy was described (540) for controlling bioﬁlms through generation of hydrogen peroxide at the bioﬁlm-surface interface rather than simply applying a disinfectant extrinsically.. tuberculosis (4. (225) concerning the survival of B.. Bioﬁlms provide the most important example of how physiological (phenotypic) adaptation can play a role in conferring intrinsic resistance (57)...... coli and Salmonella and includes the production of neutralizing enzymes to prevent cellular damage (including peroxidases.... Slow-growing bacteria are particularly insusceptible.. (iv) production of degradative enzymes (and neutralizing chemicals)... The development of adherent microcolonies is thereby initiated. For example..... In the studies by Anderson et al...Associated with enhanced tolerance to antiseptics and disinfectants?
notably biocorrosion.... Legionella pneumophila is often found in hospital water distribution systems and cooling towers..... contaminating organisms may be less susceptible to this treatment (140). which compromised subsequent sterilization with glutaraldehyde.. 98)... Bacteria in different parts of a bioﬁlm experience different nutrient environments... the permeability agent EDTA has only a temporary potentiating effect in the catheterized bladder..... because of bioﬁlm production... In addition........ Recent nosocomial outbreaks due to M. catalases. 12– 14) concerning the contamination of iodophor antiseptics with Pseudomonas.. These outbreaks were associated with inadequate cleaning of scopes.... The so-called oxidative-stress or SOS response has been well studied in E. Subculture of these cells in routine culture media resulted in reversion to sensitivity (218).. 171. 12–14)... 539).. glutathione reductase) and to repair DNA lesions (e.. Pseudomonas bioﬁlms were found on the interior surfaces of polyvinyl chloride pipes used during the manufacture of providone-iodine antiseptics. and their physiological properties are affected (57).Depends on (i) nature of antiseptic/disinfectant... a point reiterated recently in another context (126). Increased resistance to chlorine has been reported for Vibrio cholerae.. While these organisms do not form a true bioﬁlm. 172)... 12.. the phenotypes of sessile organisms within bioﬁlms differ considerably from the planktonic cells found in laboratory cultures (73)... Within the depths of a bioﬁlm.. probably as a consequence of modiﬁcations in their outer membranes (56. reduced water quality......... (ii) binding capacity of glycocalyx toward antiseptic or disinfectant.Associated with (i) nutrient limitation and (ii) growth rate Increased production of degradative enzymes by attached cells. and (iii) rate of growth of microcolony relative to diffusion rate of chemical inhibitor Modulation of microenvironment ... Bacteria within these bioﬁlms reside in speciﬁc microenvironments that differ from those of cells grown under normal laboratory conditions and thus show variations in their response to antiseptics and disinfectants. including catalase and glutathione reductase (497)
ANTISEPTICS AND DISINFECTANTS TABLE 8.VOL... however. However... marcescens in 2% chlorhexidine solutions.... 142. M.. bacteria removed from a bioﬁlm and recultured in culture media are generally no more resistant than the “ordinary” planktonic cells of that species (57).... 59.... nutrient limitation is likely to reduce growth rates...... with bacterial growth subsequently recurring (495).... fattened cells of S... resulting in increased infection rates and possible recurrence of infection (125)... chelonae (discussed under “Intrinsic resistance of mycobacteria”)...... (ii) chemical interaction between the disinfectant and the bioﬁlm itself... which was attributed to the embedding of these organisms in a thick matrix that adhered to the walls of a storage containers. Several instances are known of the contamination of antiseptic or disinfectant solutions by bacteria.. 497). Recently... Several reasons can account for the reduced sensitivity of bacteria within a bioﬁlm (Table 8)..... increased tolerance can be obtained by pretreatment with a subinhibitory dose of hydrogen peroxide (113.. (iii) modulation of the microenvironment. Chlorination in combination with continuous heating (60°C) of incoming water is usually the most important disinfection measure.. so that eventually a continuous bioﬁlm is produced on the colonized surface...... for example. many aerobic microorganisms have developed intrinsic defense systems that confer tolerance to peroxide stress (in particular H2O2) in vivo... Planktonic cultures grown under conditions of nutrient limitation or reduced growth rates have cells with altered sensitivity to disinfectants.. marcescens of a benzalkonium chloride solution implicated in meningitis (468).. the colonized surface incorporated a catalyst that generated the active compound from a treatment agent......Mechanism unclear at present Plasmid transfer between cells in bioﬁlm?..... Sister cells then arise by cell division and are bound within the glycocalyx matrix... In this procedure...... For example... It is to be wondered whether a similar reason could be put forward for the contamination by S. The interaction of bacteria with surfaces is usually reversible and eventually irreversible.. and a glycocalyx may be associated with intrinsic resistance to the bisbiguanide
(360)... or (v) genetic exchange between cells in a bioﬁlm.. Colonization also occurs on implanted biomaterials and medical devices.... Interestingly....... Marrie and Costerton (310) described the prolonged survival of S..... aureus produced by repeated subculturing in glycerol-containing media are more resistant to alkyl phenols and benzylpenicillin than are wild-type strains (220)... 114.... In both organisms..... 323) and HCV (53) underscore the importance of pseudobioﬁlm formation in ﬂexible ﬁberoptic scope contamination... There may be (i) reduced access of a disinfectant (or antibiotic) to the cells within the bioﬁlm... Pretreatment induces a series of proteins. the cross-linking action of glutaraldehyde can cause a buildup of insoluble residues and associated microorganisms on scopes and in automated reprocessors..... Similar conclusions were reached by Hugo et al...... Gram-negative pathogens can grow as bioﬁlms in the catheterized bladder and are able to survive concentrations of chlorhexidine that are effective against organisms in noncatheterized individuals (493. Other examples are also known.. exonuclease III) (112. One can reach certain conclusions about bioﬁlms.. 494)........g. (10.. Thus.. B.... Irreversible adhesion is initiated by the binding of bacteria to the surface through exopolysaccharide glycocalyx polymers.. many of which are under the positive control of a sensor/regulator protein (OxyR). which can affect susceptibility to antimicrobial agents (98.... which expresses an amorphous exopolysaccharide causing cell aggregation (“rugose” morphology ) without any loss in pathogenicity....... cepacia freshly isolated from the hospital environment is often considerably more resistant to chlorhexidine than when grown in artiﬁcial culture media..
128. Strains of Providencia stuartii may be highly tolerant to Hg2 . 250. This compound has a much greater effect on gram-positive than gram-negative bacteria. epidermidis. Resistance to mercury is plasmid borne. “resistance. aureus. It was concluded that apart from certain speciﬁc examples such as silver. Deinococcus (528). (ii) Cell surface alterations (outer membrane proteins) Efﬂux: some S. (511). High levels of tolerance to chlorhexidine and QACs (311) may be intrinsic or may have resulted from mutation. This is particularly true with MIC analysis. The mechanism of resistance has yet to be elucidated fully but may be associated with silver accumulation (152. and neomycin and kanamycin) into E. some S. this topic was considered further by Russell (413). some S.164
MCDONNELL AND RUSSELL TABLE 9. aureus containing penicillinase plasmids (110). and hypochlorous acid has also been reported (81. 200). (492). 427) (Table 9). but Bacillus tolerance to H2O2 has been described to vary during the growth phase (127) and in mutant strains (67. often indicating that the target organism is unaffected by its antimicrobial action. 496) that the extensive usage of these cationic agents could be responsible for the selection of antiseptic-disinfectant-. (iii) Decreased uptake(?) (i) Efﬂux: some S.. however. Plasmids and bacterial resistance to antiseptics and disinfectants. 423. B. inducible. or chlorinated phenols. aureus. Increased biocide MICs due to acquired mechanisms have also been reported and in some case misinterpreted as indicating resistance. be considered in evaluating the clinical implications of these reports. and may be transferred by conjugation or transduction. members of the Enterobacteriaceae (479. For example. ethanol. REV. and organomercurials.” or an increase in the MIC of a biocide. 348.
and further nonessential proteins that accumulate to protect the cell (338). 335). All of these studies demonstrated that it was difﬁcult to transfer chlorhexidine or QAC resistance under nor-
. Organomercurials are still used as preservatives. Since then. 373. Plasmid-encoded resistance to silver has been found in Pseudomonas stutzeri (192). epidermidis
Mercurialsb Ethidium bromide
Now rarely used for antiseptic or disinfectant purposes. acquired resistance to antiseptics and disinfectants can arise by either mutation or the acquisition of genetic material in the form of plasmids or transposons. The oxidative stress response in gram-positive bacteria is less well studied. silver compounds. aureus. QACs. with resistance to the above compounds and to additional organomercurials (331). Transformation of this plasmid (which encodes resistance to carbenicillin.34% H2O2 (200). No evidence has been presented to show that there is a plasmid-linked association between antibiotic resistance and disinfectant resistance in these organisms. other metals. cationic disinfectants (such as chlorhexidine and QACs). does not necessarily correlate with therapeutic failure. (i) Plasmid-mediated antiseptic and disinfectant resistance in gram-negative bacteria. epidermidis. An increase in an antibiotic MIC can may have signiﬁcant consequences. Unlike antibiotics. Mercurials are no longer used as disinfectants. Silver salts are still used as topical antimicrobial agents (50. (ii) Decreased uptake(?) Decreased uptake. Plasmid-encoded resistance to antiseptics and disinfectants
had at one time been most extensively investigated with mercurials (both inorganic and organic). bactericidal activity. some S. so that it is difﬁcult to assess the signiﬁcance of this ﬁnding. It is important to note that “resistance” as a term can often be used loosely and in many cases must be interpreted with some prudence. Acquired Bacterial Resistance Mechanisms As with antibiotics and other chemotherapeutic drugs. no inactivation (cf.
Antiseptics or disinfectants
Chlorhexidine salts QACs Silver compounds Formaldehyde Acridinesa Diamidines Crystal violeta
(i) Inactivation: not yet found to be plasmid mediated. 518). Inorganic mercury (Hg2 ) and organomercury resistance is a common property of clinical isolates of S. and triclosan. epidermidis Inactivation (reductases. Sutton and Jacoby (498) observed that plasmid RP1 did not signiﬁcantly alter the resistance of P. epidermidis Efﬂux: some S. and antibiotic-resistant strains. however. It is important that issues including the pleiotropic action of most biocides. and Haemophilus inﬂuenzae (36). aeruginosa did not increase the sensitivity of these organisms to a range of antiseptics (5). Possible mechanisms of plasmid-encoded resistance to antiseptics and disinfectants
Chemical agent Examples Mechanisms
CLIN. High levels of disinfectant resistance have been reported in other hospital isolates (195). aureus. coli or P. MICROBIOL. and the topic was reconsidered (83. aeruginosa to QACs. some S. tetracycline. 480. subtilis mutants have been described to be more resistant at 0. (ii) efﬂux: some S. Occasional reports have examined the possible role of plasmids in the resistance of gram-negative bacteria to antiseptics and disinfectants. as well as to diamidines. or broad-spectrum. acridines and ethidium bromide. formulation effects. Similar inducible defense mechanisms were described for Campylobacter jejuni (185). although increased resistance to hexachlorophene was observed. chlorhexidine. mercury compounds) (i) Inactivation by formaldehyde dehydrogenase. some S. there have been numerous reports linking the presence of plasmids in bacteria with increased tolerance to chlorhexidine. plasmids were not normally responsible for the elevated levels of antiseptic or disinfectant resistance associated with certain species or strains. Chopra (82. etc. concentrations used in products. Cross-resistance to heat. and Citrobacter spp. aureus. specifying resistance to Hg2 and to some organomercurials.5% H2O2 than are wild-type strains at 0. some S. although no clear-cut role for plasmid-speciﬁed resistance has emerged (102. 83) examined the role of plasmids in encoding resistance (or increased tolerance) to antiseptics and disinfectants. epidermidis Efﬂux: some S. and other cations and anions. there is little evidence to support this conclusion. aureus. or Proteus spp. but phenylmercuric salts and thiomersal are still used as preservatives in some types of pharmaceutical products (226). the level of increased tolerance to H2O2 during the oxidative stress response may not afford signiﬁcant protection to concentrations used in antiseptics and disinfectants (generally 3%). 443). chromosomally mediated inactivation. However. It has been proposed (492. and antibiotics (496). 511). iodine. direct product application. pseudomonads. lyases) Efﬂux: some S. Plasmids conferring resistance to mercurials are either narrow spectrum. 511).
aureus strains to some antiseptics and disinfectants
qac genea MIC ratiosb of c: Proﬂavine CHG Pt Pi CTAB BZK CPC DC
ANTISEPTICS AND DISINFECTANTS
qacA qacB qacC qacD MIC ( g/ml) for sensitive strain
16 8 1 1 40
2. aureus strains were 100 and
TABLE 11. Genes encoding increased QAC tolerance may be widespread in food-associated staphylococcal species (203). the qacC gene may have evolved from qacD (288). cetyltrimethylammonium bromide. aureus carrying various qac genes divided by the MIC for a strain carrying no gene (the actual MIC for the test strain is shown in the bottom row).VOL. 1999 TABLE 10. aureus and CNS have a common pool of resistance determinants. CPC. pentamidine isethionate. cepacia (476). aeruginosa. ethidium bromide
The qacK gene has also been described. especially P. These biocidal agents include chlorhexidine. dequalinium chloride. Increased antiseptic MICs have been reported to be widespread among MRSA strains and to be speciﬁed by two gene families (qacAB and qacCD) of determinants (188. b Calculated from the data in reference 289. c CHG. ethidium bromide QACs. The qacB gene is detected on large heavy-metal resistance plasmids. A gene encoding multidrug resistance was not found in susceptible strains but was present in 70% of S. 368. with a possible selective advantage in possessing both as opposed to qacA only (281). It has been known for several years that some antiseptics and disinfectants are. plasmid R124 alters the OmpF outer membrane protein in E. These genes have identical target sites and show restriction site homology. However. In S. (ii) Plasmid-mediated antiseptic and disinfectant resistance in staphylococci. acridines. 289. 90). acridines. 246]) and formaldehyde dehydrogenase (247. probably by virtue of a permeability barrier (428). 289. (31) demonstrated a relationship between increased S. aureus (MRSA) strains are a major cause of sepsis in hospitals throughout the world. In addition. 281. also -lactamase and heavy-metal resistance families -Lactamase and heavy-metal resistance plasmids Small plasmids ( 3 kb) or large conjugative plasmids Large (50-kb) conjugative. and QACs. and chlorocresol) or to the preservatives known as parabens (8). benzalkonium chloride. 317). aureus strains as an integrated family plasmid or part thereof. The qacA gene is present predominantly on the pSK1 family of multiresistance plasmids but is also likely to be present on the chromosome of clinical S. Dussau et al. (129) and Behr et al. there is growing evidence that S. aureus strains that contain a plasmid carrying genes encoding resistance to the aminoglycoside antibiotic gentamicin (Table 10). CTAB. The potential clinical signiﬁcance of this ﬁnding remains to be determined. According to Sasatsu et al. Some 40% of isolates of coagulasenegative staphylococci (CNS) contain both qacA and qacC genes. ethidium bromide Some QACs. multiresistance plasmids
QACs. benzalkonium chloride. aureus is considerably higher than the normal quoted value (ca. these mechanisms are encoded by at least three separate multidrug resistance determinants (Tables 10 and 11). qac genes and resistance to quaternary ammonium compounds and other antiseptics and disinfectants
Multidrug resistance determinanta Gene location Resistance encoded to
qacA qacB qacCb qacDb
pSK1 family of multiresistant plasmids. 1 ca. but it is likely to be less signiﬁcant than qacAB in terms of antiseptic or disinfectant tolerance. on the basis of MICs. 423. Bacterial resistance mechanisms to formaldehyde and industrial biocides may be plasmid encoded (71. somewhat less inhibitory to S. coli.8
16 4 ca. chlorhexidine diacetate.
mal conditions and that plasmid-mediated resistance to these chemicals in gram-negative bacteria was an unlikely event. Patients at particularly high risk are those who are debilitated or immunocompromised or who have open sores. Methicillin-resistant S. Ratios are MICs for strains of S. however. 363–365. Pi. aureus.5 1 1 1 0. cresol. ethidium bromide Some QACs. (505) proposed that increased resistances to cetyltrimethylammonium bromide (CTAB) and to propamidine isethionate were linked and that these cationic agents may be acting as a selective pressure for the retention of plasmids encoding resistance to them. DC. Staphylococci are the only bacteria in which the genetic aspects of plasmid-mediated antiseptic and disinfectant resistant mechanisms have been described (466).000-fold) to povidone-iodine. 12. and cells containing this plasmid are more resistant to a QAC (cetrimide) and to other agents (406). 288. 238. 1 50
16 2 1 1 50d
4 2 6 6 1
3 3 3 3 2
4 2 4 4 1
2 2 1 1 4
a qac genes are otherwise known as nucleic acid binding (NAB) compound resistance genes (88). (395. the MICs of triclosan against sensitive and resistant S. Low-level transferable resistance to triclosan was reported in MRSA strains (88. Interesting studies by Reverdy et al. hexamine. 280. 295. aureus MICs to the -lactam oxacillin and four antiseptics (chlorhexidine. Triclosan is used in surgical scrubs. Tennent et al. According to Mycock (346). although not all strains have increased virulence. Pt. 463). MRSA strains showed a remarkable increase in tolerance (at least 5. diamidines. there was no decrease in susceptibility of antibiotic-resistant
strains to phenolics (phenol. 427. diamidines. no further work on these organisms has been described. 193). cetylpyridinium chloride. d The MIC of propamidine isethionate for the sensitive S. chlorhexidine salts. 269) are considered to be responsible (202). Many can be referred to as “epidemic” MRSA because of the ease with which they can spread (91. BZK. The qacC and qacD genes encode identical phenotypes and show restriction site homology. By contrast. qac genes and susceptibility of S. It is active against staphylococci and less active against most gram-negative organisms. together with ethidium bromide and acridines (8.
. (465). 506). 2 g/ml [Table 6]). The qacAB family of genes (Table 11) encodes protondependant export proteins that develop signiﬁcant homology to other energy-dependent transporters such as the tetracycline transporters found in various strains of tetracycline-resistant bacteria (405). Alterations in the cell surface (outer membrane proteins [19. soaps. 396). the so-called TOM plasmid encodes enzymes for toluene and phenol degradation in B. and acriﬂavine). propamidime isethionate. and deodorants. 367. Furthermore. aureus strains for which the MICs of all four of these antiseptics were increased and in 45% of the remaining strains resistant to at least one of these antiseptics (31).
The resistant strains did not show altered biochemical properties of changed virulence for mice. (23) have pointed out that for antibiotics. (463) showed that duplication of ebr is responsible for resistance to ethidium bromide and to some antiseptics. marcescens. fewer studies have been performed to determine whether mutation confers resistance to antiseptics and disinfectants. From this. two MRSA strains grew normally with a threefold increase in viable numbers over a 4-h test period whereas an MSSA strain showed a 97% decrease in viability. It was. who worked with MRSA and other strains of S. gentamicin. However. which is practically insoluble in water. Irizarry et al.400 g/ml. Most of these studies with sensitive and “resistant” strains involved the use of MIC evaluations (for example. (229) compared the susceptibility of MRSA and MSSA strains to CPC and chlorhexidine by both MIC and bactericidal testing methods. 289. (89) pointed out that curing of resistance plasmids produced a fall in MICs but not a consistent decrease in the lethal activity of chlorhexidine. chlorhexidine. stutzeri (502). aureus. Enterococcus faecium strains showing high level resistance to vancomycin. One problem associated with QACs and chlorhexidine is the turbidity produced in liquid culture media above a certain concentration (interaction with agar also occurs). Antibiotic-resistant corynebacteria may be implicated in human infections. A similar efﬂux system is present in Enterococcus hirae (326). coli or S. (229) also concluded that MRSA strains are less susceptible than MSSA strains to both chronic and acute exposures to antiseptics and disinfectants. stable chlorhexidine resistance was developed in P. (354). 11. arrived at their highly selective conclusions. the turbidities (and viability) of the two MRSA and one MSSA strains decreased at very similar rates (if anything. could adapt to grow in 100. but resistance could be lost when the cells were grown on QAC-free media.g. Similar observations were made by Cookson et al. or both are not more resistant to chlorhexidine or other nonantibiotic agents (9. Irizarry et al. but studies with plasmid-containing and plasmid-cured derivatives produced no evidence of plasmidassociated resistance (285). Recently. Sasatsu et al. and by McDonnell et al. aureus (MSSA) and MRSA strains with increased triclosan MICs (up to 1. which could undoubtedly interfere with the determination of growth. ‘Group JK’ coryneforms (Corynebacterium jeikeium) were found to be more tolerant than other coryneforms to the cationic disinfectants ethidium bromide and hexachlorophene. however. mirabilis. (319). especially in the immunocompromised. these results were disputed because these concentrations are well in excess of the solubility of triclosan (515). strains of Streptococcus mutans remain sensitive to it (235).and 258-fold increases. 289. these strains showed
. despite the extensive dental use of chlorhexidine. 319). their results with 4 g of CPC/ml show no such pattern: at this higher concentration. (318. Midgley (324. 368. therefore. A few investigations examined the bactericidal effects of antiseptics. P. one MRSA strain appeared to be affected to a slightly greater extent that the MSSA strain). Furthermore. Table 6). there is little or no evidence of plasmid-associated resistance of nonstaphylococcal gram-positive bacteria to antiseptics and disinfectants. ebr was found in antibiotic-resistant and -sensitive strains of S. Furthermore. The nucleotide sequences of the ampliﬁed DNA fragment from sensitive and resistant strains were identical. Baquero et al. aureus for which the MICs of chlorhexidine. ethidium resistance determinant from S. CNS. chlorhexidine. aureus plasmids transferred into E. in resistance to chlorhexidine. For example. demonstrated over 40 years ago (77. e. aureus.000 g of a QAC per ml. whereas it was not possible to develop resistance to chlorhexidine in Salmonella enteritidis. (148) were unable to develop stable chlorhexidine resistance in E.6 g/ml) but showed that the MBCs for these strains were identical. REV. There is a poor correlation between MIC and the rate of bactericidal action of chlorhexidine (88. aureus. (383) reported that resistance to chlorhexidine could be induced in some organisms but not in others. as well as to ethidium bromide (239. which are expelled from the cells.166
MCDONNELL AND RUSSELL
CLIN. metals (349). Furthermore. aureus cloned in E. and it was proposed that in antiseptic-resistant cells
there was an increase in the copy number of the ebr (qacCD) gene whose normal function was to remove toxic substances from normal cells of staphylococci and enterococci. 405). MICROBIOL. marcescens but not in P. Plasmid-mediated efﬂux pumps are particularly important mechanisms of resistance to many antibiotics (85). Mutational resistance to antiseptics and disinfectants. (iii) Plasmid-mediated antiseptic and disinfectant resistance in other gram-positive bacteria. 325) demonstrated that a plasmid-borne. the conclusion of this study that MRSA strains were more resistant warrants additional comments. 78) that S. Fitzgerald et al. Moreover. 319) have described methicillin-susceptible S. aureus. normally inhibited by QACs at 100 g/ml. respectively. Sasatsu et al. these results were not surprising. and butylparaben were the same as for a low-level resistant strain. diamidines. and some strains were resistant to the QAC benzalkonium chloride. MRSA strains were some four times more resistant to chlorhexidine and ﬁve times more resistant to a QAC (CPC) than were MSSA strains. To date. it was proposed that qacA and related genes carrying resistance determinants evolved from preexisting genes responsible for normal cellular transport systems (405) and that the antiseptic resistance genes evolved before the introduction and use of topical antimicrobial products and other antiseptics and disinfectants (288. the MIC quoted for methylparaben comfortably exceeds its aqueous solubility. However. Whether low-level resistance to cationic antiseptics. respectively. the presence of a speciﬁc resistance mechanism frequently contributes to the long-term selection of resistant variants under in vivo conditions. Despite extensive experimentation with a variety of procedures. At a concentration in broth of 2 g of CPC/ml. Sasatsu et al. This observation is reinforced by the ﬁndings presented by Nicoletti et al. 363–368). 89.
6. mirabilis and S. Cookson et al. 319). QACs. The evidence is currently contentious and inconclusive. Prince et al. Based on DNA homology. and enterococcal strains. McDonnell et al. On the basis of rather high actual MICs. who worked with MRSA and enterococci. CTAB. considering that biocides (unlike antibiotics) have multiple cellular targets. Chromosomal mutation to antibiotics has been recognized for decades. coli are responsible for conferring increased MICs of cationic agents to the gram-negative organism (505. can likewise provide a selective advantage on staphylococci carrying qac genes remains to be elucidated. 319) and triclosan (90. the authors stated that chlorhexidine gave similar results to CPC. (466) examined the origin of ebr (now known to be identical to qacCD) in S. Later. Recombinant S. marcescens displayed 128. 365. (464) described a high-level resistant strain of S. By contrast. 20. The resistant and sensitive cells had different surface characteristics (electrophoretic mobilities). the authors concluded that it was reasonable to speculate that the residual amounts of antiseptics and disinfectants found in the hospital environment could contribute to the selection and maintenance of multiresistant MRSA strains. It is therefore difﬁcult to see how Irizarry et al. 324–336. and acridines.. resistance to chlorhexidine was stable in S. 544). and cationic disinfectants and antiseptics such as QACs. coli encodes resistance to ethidium bromide and to QACs. 367. (89).
Moken et al. coli to chloroxylenol and to a QAC. It has been proposed (270. resistance of P. thereby limiting uptake of antiseptics and disinfectants. 240). At present.. and some antibiotics. coli mutants that overexpressed MarA and demonstrated low levels of cross-resistance to antibiotics. (333) have found that low concentrations of pine oil (used as a disinfectant) could select for E. Outer membrane modiﬁcation is an obvious factor and has indeed been found with QAC-resistant and amphoteric compound-resistant P. ﬂuorescens to QACs was reduced when EDTA was present with the QAC (although the lethal effect was mitigated after the cells were grown in medium containing QAC and EDTA). Although such systems are most important in the context of antibiotic resistance. Subinhibitory antibiotic concentrations may cause subtle changes in the bacterial outer structure. The adaptation of P. 330. 351) that proﬂavine-sensitive and -resistant cells are equally permeable to the acridine but that resistant cells possessed the ability to expel the bound dye. 319). the cell wall presents a barrier to reduce or exclude the entry of an antimicrobial agent. Possible mechanisms of fungal resistance to antiseptics and disinfectants
Type of resistance Possible mechanism Example(s)
various levels of increased tolerance to QACs. The significance of these and other MDR systems in bacterial susceptibility to antiseptics and disinfectants. but the available information links cell wall glucan. EDTA has long been known (175. It is also pertinent to note here the recent ﬁndings of Langsrud and Sundheim (274). 279. in this instance to explain acridine resistance. Another insusceptibility mechanism has been put forward. Gale (165) demonstrated a phenotypic increase in the resistance of Candida albicans to the polyenic antibiotic amphotericin B as the organisms entered the stationary growth phase. and (ii) acquired resistance. it remains to be tested if residual concentrations of antiseptics or disinfectants in clinical situations could produce the same effect. 209. Hospital (as for other environmental) isolates of gram-negative bacteria are invariably less sensitive to disinfectants than are laboratory strains (196. culture age inﬂuences the response of S. wall thickness. In this study. The evidence to date is somewhat patchy. the development of resistance is associated with changes in the cell envelope. probably as a result of a nonspeciﬁc alteration of the cell envelope (452). 12. This implies that the mechanism of such resistance is nonspeciﬁc and that it involves cellular changes that modify the response of organisms to unrelated biocidal agents. the E. with cross-resistance to a QAC. and relatively porosity to the susceptibility of Saccharomyces cerevisiae to chlorhexidine (Table 13) (204–208). outer membrane proteins. especially pseudomonads. any factor increasing glucanase activity increased amphotericin sensitivity. The adaptation and growth of S. coli of the RobA protein that confers multiple antibiotic and heavy-metal resistance (interestingly. and detergents. in particular the issue of cross-resistance with antibiotics. have been described previously (166). perhaps more importantly. Resistance to amphoteric surfactants has also been observed. 410) to produce changes in the outer membrane of gram-negative bacteria. very little is known about the ways in which fungi can circumvent the action of antiseptics and disinfectants (104. 89. Additionally. increased the susceptibility of E. 492). Protoplasts of this organism prepared by glucuronidase in the presence of -mercaptoethanol are lysed by chlorhexidine concentrations well below those effective against “normal” (whole) cells. must be studied further. which was attributed to cell wall changes involving tighter cross-linking (74). coli mutants for which the MICs of triclosan were increased (318). again. Ag may be efﬂuxed ). dyes. and (iii)
Exclusion Enzymatic inactivation Phenotypic modulation Efﬂux Mutation Inducible efﬂux Plasmid-mediated responses
Chlorhexidine Formaldehyde Ethanol Not demonstrated to datea Some preservative Some preservativesa Not demonstrated to date
a Efﬂux is now known to be one mechanism of fungal resistance to antibiotics (531). selection and mutation could play an important role in the presence of these isolates. coli to pine oil. 2. Chloroxylenol-resistant strains of P. Furthermore.g.
the MarA protein controls a set of genes (mar and soxRS regulons) that confer resistance not only to several antibiotics but also to superoxide-generating agents. multidrug resistance (MDR) is a serious problem in enteric and other gram-negative bacteria. and because they are distributed ubiquitously. Furthermore. interestingly. taking up much less [14C]chlorhexidine gluconate (206). it is difﬁcult to translate these laboratory ﬁndings to actual clinical use. e. aeruginosa (240) and with chlorhexidine-resistant S. 355). aeruginosa to QACs is a well-known phenomenon (1. These genes are activated by induction or mutation caused by some types of stress. Since plasmidmediated transfer has apparently been ruled out (see above). a natural property or development of an organism (201). but not of mar. it appears that. 111. This is an important point and one that has been reinforced by more recent studies that demonstrate the signiﬁcance of efﬂux in resistance of bacteria to antibiotics (284. similar results have been found with laboratory-generated E. In addition. 1999
ANTISEPTICS AND DISINFECTANTS TABLE 12. cerevisiae to chlorhexidine. 88. thereby stimulating cell-to-cell contact (109). but the resistance was unstable (432). In one form of intrinsic resistance. 296). There are two general mechanisms of resistance (Table 12): (i) intrinsic resistance. genetic transfer is not needed. Deletion of the mar or acrAB locus (the latter encodes a PMF-dependant efﬂux pump) increased the susceptibility of E. deletion of acrAB. Thus. The porosity of the yeast cell wall is affected by its chemical
. aeruginosa were isolated by repeated exposure in media containing gradually increasing concentrations of the phenolic. marcescens (166).VOL. coli more sensitive to hydrophobic antibiotics. the cells walls are much less sensitive at stationary phase than at logarithmic growth phase (208). and some studies have demonstrated that antibiotic-resistant bacteria are not signiﬁcantly more resistant to the lethal (or bactericidal) effects of antiseptic and disinfectants than are antibiotic-sensitive strains (11. marcescens in contact lens disinfectants containing chlorhexidine. (i) mutations at an acr locus in the Acr system render E. triclosan. Such changes involve fatty acid proﬁles and. George (168) provides several examples of MDR systems in which an operon or gene is associated with changes in antiseptic or disinfectant susceptibility. MDR is a term used to describe resistance mechanisms used by genes that form part of the normal cell genome (168). cross-resistance to chlorhexidine has been noted (240). and. Mechanisms of Fungal Resistance to Antiseptics and Disinfectants In comparison with bacteria. coli MdfA (multidrug transporter) protein was recently identiﬁed and confers greater tolerance to both antibiotics and a QAC (benzalkonium) (132). 286. (ii) the robA gene is responsible for overexpression in E.
.... repeated exposure of E. although more
QACs Benzalkonium chloride Cetrimide/CTAB Chlorhexidine
10 25 20–40
100–200 100 400
100–200 250 200
Derived in part from data in reference 525. but only a few studies with this organism and with molds have been performed... Hiom et al... Cells with linoleic acid-enriched plasma membranes are more resistant to ethanol than are cells with oleic acid-enriched ones.. Mechanisms of Viral Resistance to Antiseptics and Disinfectants Early studies on the effects of disinfectants on viruses were reviewed by Grossgebauer (189). REV. because they possessed a lipid envelope (e.. C... Molds are generally more resistant than yeasts (Table 14) and considerably more resistant than nonsporulating bacteria (Table 15)... and C (other nonlipid viruses larger than those in group B) and disinfectants into two groups...g... such as 2-phenylphenol.... cerevisiae to chlorhexidine.. hypochlorite.19.. coli phage f2 to chlorine was claimed to increase its resistance to disinfection (542). 0... B (nonlipid picornaviruses). and hydrogen peroxide.. which contains lipids and is a typical unit membrane. 2-.... (117–119) used the uptake of ﬂuorescein isothiocyanurate (FITC) dextrans and the periplasmic enzyme invertase as indicators of yeast cell wall porosity.. 436). which is principally protein in nature... Thus.... and 0.. respectively (206).... It is disappointing that so few rigorous studies have been performed with yeasts and molds and antiseptics and disinfectants (see also Miller’s  treatise on mechanisms for reaching the site of action).......168
MCDONNELL AND RUSSELL TABLE 13. cerevisiae show a similar degree of sensitivity to chlorhexidine as the parent strain (204). cationic surfactants (QACs)... from which it has been inferred that a more ﬂuid membrane enhances ethanol resistance (6). but this effect is overcome by -mercaptoethanol (119). A (lipid containing). the penetration of antiseptics and disinfectants into different types of viruses and their interaction with viral components have been little studied...25.
.. glucanase-soluble ones: the latter limit cell wall porosity (119).. Thurman and Gerber (509.. As the age of an S.. Thus.Increases in cells of older cultures: reduced chlorhexidine uptake responsible for decreased activity(?) Relative porosity . A new assay for determining the relative cell wall porosity in yeast based upon polycation-induced leakage of UV-absorbing compounds was subsequently developed... sodium dodecyl sulfate-soluble mannoproteins and sodium dodecyl sulfate-insoluble..No role found to date Glucan .. are less resistant than bacterial spores to antiseptics and disinfectants (436)..
composition. broad-spectrum ones that inactivated all viruses and lipophilic ones that failed to inactivate picornoviruses and parvoviruses. Mold spores... a “two-stage”
TABLE 14.. Intact S. MICROBIOL. cerevisiae... These ﬁndings (Table 13) can provide a tentative picture of the cellular factors that modify the response of S. albicans is less sensitive and takes up less [14C]chlorhexidine overall (206). and the genome.. (206..... chlorhexidine.. although some information has been provided by investigations with bacteriophages (307)..... The mannoprotein consists of two fractions. be added that destruction of the viral capsid may result in the release of a potentially infectious nucleic acid and that viral inactivation would only be complete if the viral nucleic acid is also destroyed. not investigated to date
Data from references 204 to 208 and 436... which react strongly with amino or sulfhydryl groups might possess virucidal activity. with values of 0. It must.... Lethal concentrations of antiseptics and disinfectants toward some yeasts and moldsa
Lethal concn ( g/ l) toward: Antimicrobial agentb Yeast (Candida albicans) Molds Penicillium chrysogenum Aspergillus niger
Cell wall composition Mannan. and isopropanol.... 510) pointed out that conﬂicting results on the actions of disinfectants on different virus types were often reported. Unfortunately. cetyltrimethylammonium bromide.. DeNobel et al... Capsid proteins are predominantly protein in nature. the capsid. There is no evidence to date of antiseptic efﬂux (although benzoic acid in energized cells is believed to be eliminated by ﬂowing down the electrochemical gradient ) and no evidence of acquired resistance by mutation (except to some preservatives ) or by plasmid-mediated mechanisms (426. ethylene oxide... Klein and Deforest (259) further classiﬁed viruses into three groups (Table 16). 208) found that the relative porosity of cells decreases with increasing culture age and that there was a reduced uptake of radiolabeled chlorhexidine gluconate....Possible signiﬁcance: at concentrations below those active against whole cells. CTAB.Changes altering CHG susceptibility(?). The glucan layer is shielded from -glucuronidase by mannoproteins. for example. as well as to ether and chloroform. Lipid-enveloped viruses were sensitive to lipophilic-type disinfectants. An important hypothesis was put forward in 1963 (258) and modiﬁed in 1983 (259) in which it was proposed that viral susceptibility to disinfectants could be based on whether viruses were “lipophilic” in nature. there is a signiﬁcant increase in the cell wall thickness.31 m recorded for cells from 1-. Parameters affecting the response of S.. Bacteriophages are being considered as “indicator species” for assessing the virucidal activity of disinfectants (108) and could thus play an increasing important role in this context. Potential viral targets are the viral envelope.. chlorhexidine lyses protoplasts Cell wall thickness.. and biocides such as glutaraldehyde. cerevisiae cells were able to endocytose FITC dextrans of 70 but not of 150.. Mannan mutants of S.. and they suggested that the structural integrity of a virus was altered by an agent that reacted with viral capsids to increase viral permeability.. however.. It is tempting to speculate that the cell wall composition in molds confers a high level of intrinsic resistance on these organisms. glucan (and possibly mannoproteins) plays a key role in determining the uptake and hence the activity of chlorhexidine in S.
resistant than nonsporulating bacteria.. with the wall acting as a barrier or modulator to the entry and exit of various agents. poliovirus ).. cerevisiae culture increases. herpes simplex virus ) or “hydrophilic” because they did not (e.... cerevisiae to chlorhexidinea
Parameter Role in susceptibility of cells to chlorhexidine
CLIN. Yeasts grown under different conditions have variable levels of sensitivity to ethanol (176.... and 6-day old cultures..g.Decreases in cells of older cultures: reduced chlorhexidine uptake responsible for decreased activity(?) Plasma membrane ... 402)..
although the fact that crude preparations are often studied means that extraneous materials could.5
0. followed by chlorine dioxide.72 0. 1). may be more correct. Formaldehyde. 503). much remains to be learned about the mechanism of viral inactivation by and viral resistance to disinfectants. Another resistance mechanism also involves viral aggregation. 1). Finally. abnormal proteinaceous agents that appear to contain no agent-speciﬁc nucleic acid (385). and Giardia intestinalis. Mechanisms of Protozoal Resistance to Antiseptics and Disinfectants Intestinal protozoa. 309. and mature cysts were more resistant than preencystment trophozoites or preexcystment cysts. it is difﬁcult to explain the extremely high resistance of prions. in particular the ability to sporulate. With the information presently available.17 3. Prions survive acid treatment. Cyst forms are invariably the most resistant to chemical disinfectants (Fig. In this context. or ineffective infection control practices.
disinfection system could be an efﬁcient means of viral inactivation while overcoming the possibility of multiplicity reactivation (ﬁrst put forward by Luria ) to explain an initial reduction and then an increase in the titer of disinfectanttreated bacteriophage. 1999
ANTISEPTICS AND DISINFECTANTS TABLE 15.4 —d —d
13. it is apparent that they could play a signiﬁcant role in modulating the effects of chemical agents. According to Taylor (503). there remains the possibility of viral adaptation to new environmental conditions.66 1.001 0. Entamoeba histolytica. take up fewer disinfectant molecules from solution than do vegetative forms. adaptation of pseudomonads. save to comment that the protease-resistant protein is abnormally stable to degradative processes. mask the true efﬁcacy of these agents (503). are all potentially pathogenic to humans and have a resistant. Many of these reports of resistance have often paralleled issues including inadequate cleaning.1 6. such as Cryptosporidium parvum.1
0. and the protective effects of bioﬁlms.06 —c
—c 0. the most signiﬁcant are clearly intrinsic.67
Abstracted from the data in references 244 and 245.1 6. (28) described the development of poliovirus having increased resistance to chlorine inactivation. “resistance” may be incorrectly used and “tolerance. iodine. thereby presenting a classical type of intrinsic resistance mechanism
(163). Acanthamoebae are capable of forming bioﬁlms on surfaces such as contact lenses (186). Of the mechanisms that have been studied.VOL. Some acquired mechanisms (in particular with heavy-metal resistance) have also been shown to be clinically signiﬁcant. Some recent studies have compared the responses of cysts and trophozoites of Acanthamoeba castellanii to disinfectants used in contact lens solutions and monitored the development of resistance during encystation and the loss of resistance during excystation (251–255).002
20 32. and free chlorine. A typical biphasic survival curve of enterovirus and rotavirus exposed to peracetic acid is also indicative of the presence of viral aggregates (198). some phenols. Inactivation was so rapid that the D-values could not be measured. Mechanisms of Prion Resistance to Disinfectants The transmissible degenerative encephalopathies (TDEs) form a group of fatal neurological diseases of humans and other animals. incorrect product use. An abnormal protease-resistant form (PrPres) of a normal host protein is implicated in the pathological process. sodium hydroxide.1 5.9 3. similar to spores. noninfectious virions from which complementary reconstruction of an infectious particle occurs by hybridization with the gene pool of the inactivated virions (298). but it would be reasonable to assume that cysts. D-values are the times to reduce the viable population by 1 log unit. 234). transmissible cyst (or oocyst for Cryptosporidium) (233. Prions are considered highly resistant to physical and chemical agents (Fig. ozone is the most effective protozoan cysticide. the persistence of infectivity of formaldehyde-treated poliovirus (458) and the resistance of Norwalk virus to chlorination (249). Although protozoal bioﬁlms have yet to be studied extensively in terms of their response to disinfectants.5 0.12 0.5 18.g. TDEs are caused by prions. at least to some extent. 264). CONCLUSIONS It is clear that microorganisms can adapt to a variety of environmental physical and chemical conditions. and guanidine thiocyanate are more effective (141.. and ethylene oxide have little effect on infectivity. Kinetic approach: D-values at 20°C of phenol and benzalkonium chloride against fungi and bacteriaa
Concn (%.” deﬁned as developmental or protective effects that permit microorganisms to survive in the presence of an active agent.9 9. unbuffered glutaraldehyde (acidic pH). e. and it is therefore not surprising that resistance to extensively used antiseptics and disinfectants has been reported.5 0. all of which are more effective than the chloramines (234. It is envisaged as consisting of random damage to the capsid protein or nucleic acid of clumped. 12.94 1. Bates et al. The lethal effects of chlorhexidine and of a polymeric biguanide were time and concentration dependent. Of the disinfectants available currently. d No inactivation: fungistatic effect only. but a synergistic effect with autoclaving plus sodium hydroxide treatment is observed. which cannot be underestimated. In these cases. but in most cases the results have been spec-
. there is currently no known decontamination procedure that will guarantee the complete absence of infectivity in TDE-infected tissues processed by histopathological procedures. wt/vol)
D-value (h)b against: Aspergillus niger Candida albicans Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus
Phenol Benzalkonium chloride
a b c
5. Multiplicity reactivation as a mechanism of resistance was supported by the observation of Young and Sharp (546) that clumping of poliovirus following partial inactivation by hypochlorite signiﬁcantly increased the phage titer. Clearly.01 0. The cyst “wall” appeared to act as a barrier to the uptake of these agents. although chlorine-releasing agents (especially hypochlorites).66 5. The reasons for this are unknown.05
Russell. 1990. W.. 21:1058–1063. Public Health 80:17–21. 1957. Alqurashi. 7. W. but it is questionable if the observed increased MICs of biocides could have clinical implications for hard-surface or topical disinfection (423. any possible clinical signiﬁcance of this remains to be tested. M. 26. Resistance of Pseudomonas to quaternary ammonium compounds. Appl. rabies virus. Adler-Storthz. R.. Carr. J. H. Infect. M. They are associated with the more efﬁcient use of these agents clinically and with the potential design of newer.170
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CLIN. L. Public Health Laboratory. Rousseaux. changes in the MICs have actually been shown not to be signiﬁcant (9. FEMS Microbiol. 1997. Sehulster. L. certain active agents are clearly more efﬁcacious against gram-positive than gram-negative bacteria. Ferrer. Resistance of Pseudomonas to quaternary ammonium compounds. MAC. 22:91–116. F. Melnick. L. M. D. Chlorhexidine sensitivity of Enterococcus faecium resistant to vancomycin. N. A rapid method of evaluating permeabilizing activity against Pseudomonas aeruginosa. Control Hosp. R. Newboult. 10:443–446. 38:745. Carr. L. difﬁcile (despite the intrinsic resistance of spores) may be effectively controlled physically by adequate cleaning with QAC-based products. R. J. B. Anderson. Dreesman. a particular antiseptic or disinfectant product may be better selected (as part of infection control practices) based on particular circumstances or nosocomial outbreaks. Susceptibility of some strains of enterococci and streptococci to antibiotics and biocides. J. N. L. 1971. General mechanism for the bacterial toxicity of hypochlorous acid: abolition of ATP production.. Lett. Infect. Bailly. 3:849–853. Development of poliovirus having increased resistance to chlorine inactivation. Laveran. 3:89–94. Effect of alkaline glutaraldehyde on hepatitis B virus antigens. Although signiﬁcant progress has been made with bacterial investigations. 15. For information on the inactivation of poliovirus.. Sutherland. 1989. R. Rolla. W. Environ. England.Y. more effective compounds and products.. F. and D. Microbiol.. B):5–11. and C. Anderson. D. W. Environ. Anderson. 111–132. Iodophor antiseptics: intrinsic microbial contamination with resistant bacteria. Microbiol. Resistance and structure of spores of Bacillus subtilis. W. 11. R. Patron. 1996. W. 1990. II. Infect. I. R. see reference 514. Microbiology 142:1249–1254. Russell. Carr. 56:3598–3600. M. With growing concerns about the development of biocide resistance and cross-resistance with antibiotics. REV. Bond. 13. Appl. Wheeler. 12. D. 19. J. R. Lett. see also reference 444. S. J. C. which can increase product efﬁcacy. Glutaraldehyde-based disinfectants. 21: 717–719. Efﬂux mechanisms are known to be important in antibiotic resistance. Ahonkhai. D. 17:149–151. J. C. Am. Viral classiﬁcation and response to some disinfectantsa
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S R R
S S S
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