Journal of Applied Microbiology Symposium Supplement 2002, 92, 16S–27S

Bacterial target sites for biocide action J.-Y. Maillard School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK

1. 2. 3. 4.

Summary, 16S Introduction, 16S Micro-organisms and biocides, 17S Mechanisms of action of biocides: overall target sites, 18S 4.1 Bacterial inactivation and mechanisms of action: the g-value, 18S 4.2 Biocide–micro-organism interactions, 19S 4.2.1 Interactions with outer cell components, 19S 4.2.2 Interactions at the cell cytoplasmic membrane level, 20S 4.2.2.1 Disruption of the cytoplasmic membrane, 20S 4.2.2.2 Dissipation of the proton motive force, 21S 4.2.2.3 Interactions with other enzymatic systems, 21S

1. SUMMARY Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the g-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less

4.2.3

Interactions with cytoplasmic constituents, 21S 4.2.3.1 Interactions with cell nucleic acids, 21S 4.2.3.2 Interactions with cell ribosomes, 22S 4.2.3.3 Interactions with other cell constituents, 22S 5. Mechanism of action of biocides: random detrimental effects or specific target sites?, 22S 5.1 Random detrimental effects to the bacterial cells, 22S 5.2 Specific target sites, 23S 5.3 Biocide concentration: a key issue, 23S 6. Conclusions, 23S 7. References, 24S

documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance. 2. INTRODUCTION

Correspondence to: J.-Y. Maillard, School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK.

Biocides have been used for centuries mainly as preservative agents for water (e.g. use of copper and silver vessels) and Ó 2002 The Society for Applied Microbiology

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

foodstuffs (e.g. salting and use of natural spices) but also for the art of mummification which relied, in part, on balsams. Various agents with antimicrobial properties (e.g. vinegar, wine, honey and mercuric chloride) have been used for wound dressings. The development of antiseptic surgery in the 19th century saw the introduction of disinfectant usage. The use of chlorine water, wood and coal tar and pure phenol solution expanded considerably to control the spread of infection in hospitals. A number of chemicals introduced at this period, e.g. wood tar, mercuric chloride, copper sulphate, hydrogen peroxide and chlorine-releasing agents, are still used today as biocides. Other phenolics, quaternary ammonium compounds (QACs) and chlorhexidine have been introduced more recently (Hugo 1999). Biocides are heavily used to preserve foodstuffs and medicine for both longevity of preparations (food and pharmaceutical products) and to maintain sterility (pharmaceutical products). Investigation of the antimicrobial activity of new preservatives, especially from natural products (e.g. plant extracts), has recently regained momentum. Biocides used as antiseptics and/or disinfectants are highly important to achieve appropriate levels of disinfection and sterility of surfaces and materials especially in the hospital environment. Furthermore, public awareness of microbial contamination and hygiene has driven the use of biocides in the home environment with numerous products containing biocides, such as chopping boards, washing powder, etc. The antimicrobial efficacy of biocidal formulations used for a wide range of applications (i.e. home, hospital and industrial) is generally well documented (Hugo and Russell 1999) and there is a better understanding of the factors influencing their activity (Russell 1999). However, the mechanisms of action of biocides have only been studied comparatively recently and the amount of information available is limited, although increasing. Understanding the mechanisms of action of biocides, together with the factors influencing their activity, has become a key issue for a better usage of biocidal formulations and control of the emergence of resistant micro-organisms. The objective of this paper is to review the mechanisms of antibacterial action of biocides with a particular interest in possible specific target sites. 3. MICRO-ORGANISMS AND BIOCIDES Biocide activity varies greatly between different types of micro-organisms and it might also differ between different strains of the same species. The classification of microbial susceptibility to biocides (Fig. 1), revisited by Favero and colleagues (Favero and Bond 1991), provides a useful guide for choosing appropriate biocides for a specific application. However, this classification does not attempt to explain how various micro-organisms react to biocides.

17S

Fig. 1 Classification of micro-organisms according to their sensitivity to biocides. CJD, Creutzfeld–Jacob disease agent; BSE, bovine spongiform encephalopathy agents; MAI, Mycobacterium avium intracellulare; HIV, human immunodeficiency virus. Adapted from Russell et al. (1997)

Among vegetative bacteria, mycobacteria are probably the most resistant to disinfection, followed by Gram-negative bacteria, Gram-positive bacteria being the most sensitive. Although this summarized classification mainly relates to a lack of penetration of biocides due to the composition and structure of the cell and outer walls of these organisms, it is not based on differences in bacterial target sites. The majority of bacteria fall within the general dimension of 0Æ74–4 lm. Bacteria have unicellular structures which may occur as cylindrical or spherical shapes although some cylindrical forms may present single or many twists. Appendages, pili and/or flagella can also be attached to the bacterial cell. Bacteria can also be surrounded by a capsule and slime, secreted materials that accumulate outside the cell and might play a role in bacterial insusceptibility to biocides. The fundamental divisions of the bacterial cell, i.e. cell wall, cytoplasmic membrane and cytoplasm, occur in all species. The basic structure and composition of the cytoplasm and cytoplasmic membrane are largely conserved between different types of bacteria, although subtle differences may occur. In terms of susceptibility to biocides, the structure and composition of the outer envelope are more

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

18S J . - Y . M A I L L A R D

interesting as they differ widely between micro-organisms (Fig. 2). It is generally accepted that the bacterial outer envelope is responsible for the different microbial responses to biocidal challenges. For example, the outer membrane of Gram-negative bacteria (Nikaido and Vaara 1985; Gilbert et al. 1990) and the cell wall of mycobacteria (Trias and Benz 1994) act as permeability barriers and are responsible for the intrinsic resistance of these micro-organisms to antimicrobial compounds. These structures are of prime

importance in understanding the efficacy and mechanisms of action of biocides. Some chemical agents, such as permeabilizers (e.g. ethylenediamine tetraacetic acid) or cross-linking agents (e.g. glutaraldehyde (GTA)), interact with these structures. The cytoplasmic membrane is composed essentially of a phospholipid bilayer with embedded proteins. It is semipermeable and regulates the transfer of solutes and metabolites in and out of the cell cytoplasm. It is also associated with several important enzymes involved in various cell metabolic functions (Singer and Nicholson 1972; Salton and Owen 1976). The cytoplasmic membrane is often considered as the major target site for biocides. Damage to the membrane can take several forms: (i) physical disruption of the membrane; (ii) dissipation of the proton motive force (PMF) and (iii) inhibition of membrane-associated enzyme activity. Bacterial cytoplasm contains the cell nucleic acid, ribosomes and various enzymes with different functions. These cell components are probably not primary target sites, since a biocide has to penetrate within the cell to reach cytoplasmic constituents. 4. MECHANISMS OF ACTION OF BIOCIDES: OVERALL TARGET SITES Biocides vary greatly in their chemical structures. The precise mechanism(s) of action often reflects this diversity, although the final damage, when high or lethal concentrations are used, may show considerable similarity. 4.1 Bacterial inactivation and mechanisms of action: the g-value

Fig. 2 Structure of the outer cell wall of (a) Gram-negative bacteria; (b) Gram-positive bacteria and (c) mycobacteria. Adapted from Maillard and Russell 2000

The kinetic of bacterial inactivation by biocides often departs from a true linear log survival/time event. The observation of the initial lag phase may reflect a number of events that occur before the cells begin to die. The concentration exponent (g) measures the effect of concentration on the antimicrobial activity of a chemical agent and can be calculated from survival data (Russell and Chopra 1996). Biocides with a high g-value (e.g. alcohols and phenolic compounds) are affected considerably by changes in concentration, whereas those with a low g-value (e.g. organomercurials and formaldehyde) are influenced to a lesser extent. A limited amount of information on the mechanisms of action of chemical agents can be obtained from the value of the concentration exponent. It can be observed that biocides with an g-value < 2 interact strongly with their target by chemical or ionic binding, although their mode of action may differ markedly. These include biocides that react with thiol groups in enzymes and proteins (e.g. organomercurials and silver compounds), oxidizing agents

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

(e.g. peroxygens), DNA reactors (e.g. acridines) and membrane-active agents (e.g. chlorhexidine and QACs). Biocides that possess an g-value > 4 have a weak physical interaction with lipophilic components of the bacterial cell envelope. They include phenolics, aliphatic and aromatic alcohols that act as cytoplasmic membrane disrupters and proton conductors. Chemical agents with intermediate g-values of 2–4 (e.g. organic acids and esters) might be considered to have both chemical and weakly physical actions (Hugo and Denyer 1987). 4.2 Biocide–micro-organism interactions A biocide must reach and interact with its microbial target site(s) to be effective. The penetration of a chemical agent within the cell might be an important condition for most, but not all, biocides. For example, GTA forms cross-linkage as it reacts strongly with proteins, probably inhibiting normal enzymatic activity or ‘fixing’ other structures, inhibiting important survival functions. Other less reactive biocides probably have a more subtle effect and need to penetrate inside the bacterial cells to be active. The initial reaction of a biocide with a microbial cell involves an initial binding to the cell surface, although target sites might be found within the cell. Subsequently, changes to the outer cell layer may occur to allow a biocide to penetrate the cell and reach its target site(s). Alteration of the bacterial structure at the outer layer, cytoplasmic membrane or within the cell cytoplasm may contribute to the bactericidal or bacteriostatic effect of the chemical agent, depending upon the extent of damage and the nature of the target site(s). The overall mechanism(s) of action of a biocide may be defined according to the bacterial structure against which it has its main activity. Thus, three levels of interaction can be described: (i) interaction with outer cellular components; (ii) interaction with the cytoplasmic membrane and (iii) interaction with cytoplasmic constituents. However, it is possible that a biocide acts at one or all three levels of interaction with the bacterial cells to produce its antimicrobial activity. 4.2.1 Interactions with outer cell components. Several biocides can interact with outer cellular components, although cell viability might not be affected. One of the noticeable effects of biocidal interaction with the bacterial cell is a change in cell hydrophobicity. For example, it has been observed that the hydrophobicity of Gram-negative bacteria was altered when challenged with cationic compounds, such as chlorhexidine and benzalkonium chloride (a QAC; El-Falaha et al. 1985a; El-Falaha et al. 1985b; Jones et al. 1991; Fitzgerald et al. 1992). These membrane active agents are thought to damage the cell wall and outer membrane of Gram-negative bacteria and promote their

19S

own uptake so that they can reach their target site(s) at the cell cytoplasmic membrane and within the cell cytoplasm (Gilbert et al. 1990). As mentioned earlier, because of their nature, crosslinking agents such as GTA interact with the outer components of the bacterial cells. Evidence of the important interaction of GTA with bacterial cells is exemplified by studies investigating the role of GTA pretreatment in protecting cells from lysis after challenge with chemical agents (Gorman et al. 1980). In Gram-negative bacteria, GTA interacts principally with outer components of the cell, notably lipoproteins. The high reactivity of GTA with the bacterial outer components might not necessarily induce a bactericidal effect and there have been concerns that low concentrations of GTA might protect the cells from other detrimental agents. However, at high concentrations, the high degree of cross-linking produced by GTA means that bacterial cells are unable to undertake most, if not all, of their essential functions, resulting in a bactericidal effect. Ortho-phthalaldehyde (OPA), another di-aldehyde, induces less cross-linkage with the bacterial cells and the resulting increase in penetration might account for its increase in antimicrobial efficacy (Walsh et al. 1999; Simons et al. 2000). Glutaraldehyde and OPA are highly reactive molecules and their effect at the bacterial surface is most likely an important aspect of their mechanisms of action. Gram-negative bacteria are generally less sensitive to biocides than Gram-positive bacteria because of their outer membrane (Fig. 2). There is a range of compounds that act specifically against this permeability barrier. Although these permeabilizing agents might not show a strong bactericidal activity, their use might enhance the activity of biocides (Russell and Furr 1977; Broadley et al. 1995; Ayres et al. 1999). Ethylenediamine tetraacetic acid binds divalent cations, notably Mg2+, which are essential for stabilizing the strong negative charges of the core oligosaccharide chain of the lipopolysaccharide (LPS) molecules. As a result, up to 50% of these LPS are released and the non-polar phospholipids associated with the inner leaflet of the outer membrane are then exposed at the cell surface (Haque and Russell 1974; Vaara 1992). Polycations (e.g. polylysin), lactoferrin and transferrin (both iron-binding proteins), polyphosphates and certain acids produced a similar increase in Gram-negative cell permeability (Vaara and Vaara 1983a, 1983b; Ellison et al. 1988). Lactoferricin B binds rapidly to Escherichia coli and appears to lead to disruption of normal permeability functions of the cytoplasmic membrane (Bellamy et al. 1993). Other biocides might have an effect on the cell wall, although they affect other parts of the bacterial cell. For example, hypochlorite, a powerful oxidizing agent, induces lysis in Gram-negative bacteria, ostensibly by an effect on the cell wall. Low concentrations of phenol, formalin and

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

20S J . - Y . M A I L L A R D

mercuric chloride have also been shown to rapidly lyse growing E. coli, streptococci and staphylococci (Pulvertaft and Lumb 1948). Anionic detergents, such as sodium lauryl sulphate (SLS) and sarkosyl lauryl sulphate, also produce lysis of Gram-negative bacteria (Bolle and Kellenberger 1958; El-Falaha et al. 1989). The formation of long forms of bacteria following an antimicrobial treatment may also be an indication of cell wall damage (Hughes 1956). Phenol and m-cresol produce long forms of Proteus vulgaris and Klebsiella aerogenes, respectively. Dyes such as methyl green, fuchsin, methylene blue and acridines can have a similar effect (Hugo 1971). 4.2.2 Interaction at the cell cytoplasmic membrane level. The term ‘membrane active agents’ is used for antimicrobials, such as polymyxins, phenols, parabens, biguanides, QACs and alcohols, that are active at the cytoplasmic membrane level of the bacterial cell. These compounds are unlikely to have a similar effect against the cytoplasmic membrane since their chemical structure differs markedly. 4.2.2.1 Disruption of the cytoplasmic membrane. Disruption of the cytoplasmic membrane is often exemplified by the leakage of intracellular components, potassium (K+) followed by inorganic phosphates, pool of amino acids and materials absorbing at 260 nm, nucleic acids and proteins (Lambert and Hammond 1973). Leakage is best considered as a measure of the disruption of the cell permeability barrier and it might reflect a bacteriostatic effect rather than cell death. The rate and extent of leakage may depend upon the micro-organisms, the type of membrane active agents and the factors affecting the efficacy of the biocide (e.g. concentration and temperature). The study of spheroplasts and protoplasts can provide useful information since these bacteria are more sensitive to biocides and lyse after exposure to compounds that cause gross membrane damage (Hugo and Longworth 1964; Davies et al. 1968). Phenols, cresols and their chlorinated derivatives (e.g. chlorocresol and para-chloro-meta xylenol) induce leakage of intracellular materials from bacteria (Hugo and Bloomfield 1971; Joswick et al. 1971; Frederick et al. 1974; Kroll and Anagnostopoulos 1981), although phenolics might have more subtle effects within the bacterial cells as discussed below. The mode of action of the cationic agent chlorhexidine is particularly well documented. The biguanide initially causes a high rate of leakage of intracellular components but, at higher concentrations, it causes coagulation of the cytosol (Hugo and Longworth 1964, 1965; Davies et al. 1968; Davies and Field 1969; Daltrey and Hugo 1974; Fitzgerald et al. 1989). However, there might not be an obvious relationship between the amount of cell constituents released and the decrease in cell viability (Tattawasart et al. 1999). Another biguanide, polyhexamethylene biguanide (PHMB), has been shown to

interact with membrane phospholipids (Ikeda et al. 1983) and to impair the integrity of the outer membrane of Gramnegative bacteria (Broxton et al. 1984a). The extent of membrane disruption is linked with the increasing level of polymerization (Broxton et al. 1983). Although bisbiguanides damage the cytoplasmic membrane, there might be some subtle differences in their ‘fine’ mechanisms of action. Comparative studies of chlorhexidine and alexidine that differ only by the nature of the end-group substituent (i.e. an ethylhexyl instead of a chlorophenol end-group) show significant differences in their antibacterial activity. Such differences might be associated with alexidine possessing additional binding targets at the cell envelope or with alternative intramolecular interactions (i.e. phase separation and domain formation of the membrane lipids) at the cytoplasmic membrane (Chawner and Gilbert 1989a, 1989b). Quaternary ammonium compounds also induce leakage of intracellular components, which is indicative of membrane damage (Davies et al. 1968; El-Falaha et al. 1985a, 1985b; Takasaki et al. 1994a; Tattawasart et al. 1999). The rate of leakage might be higher for Gram-positive than for Gramnegative bacteria. Cetyltrimethylammonium bromide (CTAB), used at a bactericidal concentration, appears to rupture the cell membrane (Davies et al. 1968). The primary site of action of CTAB has been suggested to be the lipid components of the membrane and cell lysis is a secondary effect (Gilby and Few 1960). At lower concentrations, CTAB might have more subtle effects on the membrane. For example, CTAB used at sublethal concentrations does not lyse E. coli ML35, a mutant that is unable to transport and accumulate b-galactosides from the medium, but restores a b-galactosides carrier-mediated process (Ulitzur 1970). Although the manner in which cationic agents interact with the cell is not always clear, it is believed that chlorhexidine and QACs combine with membrane phospholipids hence causing disruption of the cytoplasmic membrane (Takasaki et al. 1994b), whereas PHMB causes domain formation of the acidic phospholipids resulting in the same effect. The mode of action of anionic agents is also associated with a disruption of the permeability barrier, thus inducing leakage of intracellular components (Bolle and Kellenberger 1958; Voss 1963; Salton 1968; El-Falaha et al. 1989). For example, low concentrations of SLS induce lysis of protoplasts of Micrococcus lysodeikticus (Gilby and Few 1960) and of spheroplasts of E. coli (Razin and Argamam 1963). The surfactant dodecylguanidine monoacetate (dodine) has been shown to rapidly cross the outer and cytoplasmic membrane of Pseudomonas syringae and to combine with cell phospholipids and proteins. Its effect results in the rapid degradation and release of RNA and cell lysis. Higher concentrations produce cytoplasm coagulation (Cabral 1991). Amphoteric agents, such as dodecyl-di(aminoethyl)-glycine and dodecylb-alanine, combine the detergent activity of anionic

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

surfactants with the bactericidal activity properties of cationic compounds. Amphoteric surfactants cause leakage of intracellular components in bacteria but their precise mechanism of action is not known. Non-ionic agents consist of hydrocarbon chains attached to a non-polar waterattracting group (e.g. polysorbates). Low concentrations affect the permeability of the outer membrane in Gramnegative bacteria (Brown 1975). Ethanol and isopropanol are membrane disrupters. Phenylethanol and phenoxyethanol also induce generalized loss of cytoplasmic membrane function (Fitzgerald et al. 1992). Ethanol causes the rapid release of intracellular components and disorganizes the membranes, probably as the result of the penetration of the solvent into the hydrocarbon part of the phospholipid bilayer (Seiler and Russell 1991). Other agents, such as organic acids and esters, may also induce leakage of intracellular components, although they also have other effects on the bacterial cells. 4.2.2.2 Dissipation of the proton motive force. The proton motive force is involved in active transport, oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in bacteria (Mitchell 1961, 1972; Hugo 1978). The PMF is expressed as a proton gradient across the cytoplasmic membrane from the interior of the cell to the outside. Organic acids and their esters, the parabens, target the PMF. For example, sorbic acid accelerates the movement of protons in E. coli from low pH media to the cell cytoplasm (Eklund 1985a). Other studies have demonstrated that lipophilic acids and parabens inhibit the active uptake of amino and oxo acids in E. coli and Bacillus subtilis. However, the efficacy of organic acids used as preservatives for pharmaceutical and food products cannot be solely explained by an activity against the PMF. Their inhibitory activity can also be caused by an acidification of the cell cytoplasm and by inhibition of some unidentified cell function. For example, acetic acid has been shown to neutralize the PMF, lower the pHi and denature proteins (Eklund 1983; Salmond et al. 1984; Eklund 1985a, 1985b). Mlynarcik et al. (1981) observed that ATP synthesis in Staphylococcus aureus was completely inhibited by three distinct chemicals, a bisquaternary ammonium salt, an amine oxide and an alkoxy phenylcarbamic acid ester. Dinitrophenol, a weak lipophilic acid, has been shown to act as an uncoupler of oxidative phosphorylation by inhibiting ATP synthesis (Simon 1953). Phenoxyethanol induces proton translocation in E. coli, but higher concentrations produce gross membrane damage (Gilbert et al. 1977). Further evidence of dissipation of the PMF has been investigated with the bisphenols fentichlor and triclosan (Bloomfield 1974) and cationic compounds, such as CTAB that has been shown to discharge the membrane potential component (Denyer and Hugo 1977).

21S

Polyhexamethylene biguanide not only causes membrane leakage but its effects are also consistent with patterns of respiratory enzyme inhibition (Broxton et al. 1984b). 4.2.2.3 Interactions with other enzymatic systems. Many proteins embedded in the cytoplasmic membrane are enzymes. Hexachlorophane, a phenolic biocide, inhibits the membrane-bound part of the electron transport chain at low concentrations. At higher concentrations, it induces leakage of intracellular contents from B. megaterium (Joswick et al. 1971; Frederick et al. 1974). Ethanol shows secondary effects in E. coli with the inhibition of DNA, RNA, protein and peptidoglycan synthesis. Other effects, such as inhibition of enzymes involved in glycolysis, fatty acid and phospholipid synthesis and solute uptake, might result directly from an ethanolinduced disruption of the membrane structure. Other biocides interact with the thiol group in proteins. The thiol groups, which derive from cysteine residues, are vital for the activity of many enzymes. Reaction with, or oxidation of, these essential groups produces cell inhibition or cell inactivation. Metals, such as copper and silver, and the element arsenic and bronopol react similarly with enzyme or protein thiol groups (Thurman and Gerba 1989; Russell and Hugo 1994; Liau et al. 1997). The activity of 1,2-benzisothiazol-3-1, 5-chloro-n-methylisothiazol-3-1 (CMIT) and N-methylisothiazol-3-1, three isothiazolones widely used as preservatives, has been associated with their interaction with thiol groups, although CMIT might also react with amines. Furthermore, CMIT induces morphological changes in treated E. coli that suggest an additional effect on DNA synthesis (Collier et al. 1990). Chlorhexidine has been claimed to be a specific inhibitor of membranebound adenosine triphosphatase, although it might not be a primary target site to the biguanide since inhibition occurs only at high concentrations (Chopra et al. 1987; Kuyyakanond and Quesnel 1992). 4.2.3 Interactions with cytoplasmic constituents. 4.2.3.1 Interactions with cell nucleic acids. Antibacterial dyes include the tryphenylmethane group (e.g. crystal violet) and the acridines. Acridines compete with protons for anionic sites on the cell surface and combine with several sites on or in the bacterial cell, the most important of which is DNA (Gittelson and Walker 1967; Silver 1967). The attachment results from the intercalation of an acridine molecule between two layers of base pairs. Quinacrine, an antimalarial drug, blocks DNA synthesis and strongly inhibits the syntheses of RNA and proteins in E. coli (Ciak and Hahn 1967) but selectively blocks RNA formation in B. cereus (Seligman and Mandel 1971). Crystal violet has also been shown to interact with nucleic acids in E. coli (Adams 1968).

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

22S J . - Y . M A I L L A R D

Alkylating agents, such as ethylene oxide and formaldehyde, affect purine nucleosides and nucleic acids because of their interaction with amino, sulphydryl and hydroxyl groups (Hoffman 1971; Adams et al. 1981). Ozone reacts with amino acids (Prat et al. 1968; Mudd et al. 1969) and modifies purine and pyrimidine bases (Greene et al. 1993). 4.2.3.2 Interactions with cell ribosomes. Ribosomes are responsible for the translation of messenger RNA into a peptide chain. They can be damaged by biocides (e.g. hydrogen peroxide, p-chloromercuribenzoate and proflavine) although they might not be primary target sites (Miall and Walker 1966; Wang and Matheson 1967; Nakamura and Tamaoki 1968). 4.2.3.3 Interactions with other cell constituents. A number of biocides will undoubtedly react with cytoplasmic constituents other than nucleic acids and ribosomes. For example, alkylating and oxidizing agents are highly reactive chemicals that strongly interact with the bacterial cell. Ethylene oxide, propylene oxide and b-propiolactone act as alkylating agents. They react with amino, carboxyl, sulphydryl and hydroxyl groups in bacterial protein as well as nucleic acids (Hoffman 1971; Russell 1976; Adams et al. 1981). Aldehydes, especially GTA, OPA and formaldehyde, are highly reactive chemical compounds that combine with proteins (to give intermolecular cross-links) and nucleic acids (Gorman et al. 1980). Oxidizing agents, such as

halogens, hydrogen peroxide, peracetic acid and ozone, may also interact with a number of cellular constituents. Hydrogen peroxide activity results in the formation of hydroxyl radicals which oxidize thiol groups in proteins and enzymes (Turner 1983). Cationic compounds and especially chlorhexidine have been shown to cause cytoplasmic coagulation (Hugo and Longworth 1964, 1965; Davies et al. 1968; Davies and Field 1969; Daltrey and Hugo 1974; Fitzgerald et al. 1989). Other chemicals, such as the acridines, have been shown to interact with more specific target sites. For example, protein synthesis in B. megaterium is blocked by primaquine (Olenick and Hahn 1972). Proflavine inhibits the synthesis of polynucleotides by DNA polymerase of E. coli (Kadohama and McCarter 1971). Serry et al. (1986) propose that the selective inhibition of peptidoglycan and DNA synthesis by chloroacetamide (an acidamide) results in the morphological changes observed in E. coli. 5. MECHANISM OF ACTION OF BIOCIDES: RANDOM DETRIMENTAL EFFECTS OR SPECIFIC TARGET SITES? 5.1 Random detrimental effects to the bacterial cells It is generally accepted that biocides have several target sites within the bacterial cell (Table 1) and that the overall

Table 1 Bacterial targets for biocides Target site

Mechanism(s)

Biocides

GTA, OPA, FMA(?) CHA, QACs, CRAs, mercury (II) salts, PHE ACD, alcohols, anilides, CHA, QACs, PHE, HCP ACD, anilides, QACs, PHE, HCP

Interaction with specific groups

Cross-linking Increased permeability Increased permeability Membrane potential and electron transport chain Adenosine triphosphate synthesis Inhibition of enzyme activity General coagulation Nucleic acids Ribosomes Thiol groups

Biocide-induced autocidal activity

Amino groups Sulphydryl groups Accumulation of free radicals

Outer layers Cell wall Outer membrane* Cytoplasmic membrane

Cytoplasmic constituents

CHA, copper (II) salts, ETO CHA, QACs, PHE CHA, QACs, GTA, HCP, metallic salts, PHE ACD, ACR, ETO, FMA, GTA, CRAs, POP HOP, mercury (II) salts and organomercurials BOP, ETO, GTA, HOP, CRAs, IOD, POP, metallic salts, IST ETO, FMA, GTA, OPA, BOP, ETO, GTA, HOP, CRAs, metallic salts, IST BOP, IST, HOP, membrane active agents

ACD, Organic acids and parabens; ACR, acridines; BOP, bronopol; CHA, chlorhexidine; CRAs, chlorine-releasing agents; ETO, ethylene oxide; FMA, formaldehyde; GTA, glutaraldehyde; HCP, hexachlorophane; HOP, hydrogen peroxide; IOD, iodine and iodophors; IST, isothiazolones; OPA, ortho-phthalaldehyde; PHE, phenolics; POP, b-propiolactone; QACs, quaternary ammonium compounds. *Gram-negative bacteria. Copper (II) salts, mercury (II) salts and organomercurials, silver (I) salts. Agents causing damage to the cytoplasmic membrane. Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

damage leads to the bactericidal effect. This concept is certainly true for highly reactive biocides, such as alkylating and oxidizing agents. Undoubtedly, the bactericidal activity of these biocides results, at least in part, from their effect on cell cytoplasm constituents, such as cytoplasmic proteins, ribosomes and nucleic acid, although their precise target sites within the cells might be difficult to pinpoint. The precise sequence of events and target sites responsible for a bactericidal effect might also be difficult to identify. Other chemical agents, such as cationic compounds, dyes and phenolics, are less reactive and might have a selected number of interaction sites with the bacteria. The alteration of multiple sites, such as the cytoplasmic membrane and cytoplasmic constituents, is probably responsible for the bactericidal effect. For example, it is unlikely that the mode of action of chlorhexidine involves specific inhibition of a particular enzyme system, in view of the reaction of high concentrations of the biguanide with bacterial proteins (Hugo and Longworth 1966). The sequence of events leading to a bactericidal effect might be well defined. For example, the proposed sequence of events of PHMB interaction with the E. coli cell envelope is as follows: (i) rapid interaction with negatively charged bacterial surface with specific adsorption to phosphatecontaining compounds; (ii) outer membrane integrity is damaged and PHMB is attracted to the cytoplasmic membrane; (iii) PHMB binds to phospholipids and increases membrane permeability with release of K+ and (iv) complete loss of membrane function and coagulation of intracellular components leading to bactericidal effect. Furthermore, there is evidence that the transition from sublethal injury to cell death is mediated by biocide-induced self-destructive events. Dodd et al. (1997) have postulated that metabolic imbalance following biocidal challenge leads to free radical production and self-destruction. Thus, autocidal activity caused by free radical accumulation through metabolic imbalance and impaired ionic homeostasis may add to a biocidal effect (Denyer and Stewart 1998). 5.2 Specific target sites As opposed to the concept of overall damage to the bacterial cells, some studies have shown that some biocides have specific target sites within the cell. For example, it was observed that CTAB at sublethal concentrations did not lyse E. coli ML35, a mutant that is unable to transport and accumulate b-galactosides from the medium, but restored a b-galactosides carrier-mediated process (Ulitzur 1970). Pearce et al. (1999) found that sublethal concentrations of biocides had subtle effect(s) against Staph. aureus in preventing or increasing the transfer of antibiotic markers by conjugation or transduction.

23S

Several studies have shown that phenolics might have a primary target. Kranz and Lynch (1979) showed that triclosan induced pigment production in Serratia marcescens and Ps. aeruginosa by some probably direct effect on pigment metabolism. More recently, triclosan has been found to block lipid synthesis by specifically targeting the enoyl-acyl carrier protein reductase in E. coli (Heath et al. 1998; McMurry et al. 1998) and Mycobacterium smegmatis (McMurry et al. 1999). Further evidence came from structural investigations of the interaction of the bisphenol with this target site (Stewart et al. 1999). 5.3 Biocide concentration: a key issue Concentration is a major factor in biocidal activity (Russell and McDonnell 2000). Most biocide formulations contain high concentrations of active agents to achieve an optimal, broad spectrum activity for direct use on an inanimate surface, skin and in water. However, MICs of biocides are frequently quoted, although it is usually the lethal effects of a biocide that must be considered. For example, the concentrations of triclosan that inhibit enoyl reductase in E. coli and M. smegmatis are far below (Heath et al. 1998; McMurry et al. 1998, 1999) the concentrations used in formulations or in other studies (McDonnell and Pretzer 1998; Russell and McDonnell 2000). Furthermore, Rego¨s and Hitz (1974) observed that, at a low bacteristatic concentration, triclosan interfered at the cytoplasmic membrane level with the uptake of amino acids, uracils and other nutrients but, at higher bactericidal concentrations, the bisphenol caused membrane lesions that led to the leakage of intracellular contents. Minimum inhibitory concentrations have also been used to evaluate the emergence of biocide resistance in bacteria. However, the possibility of failure to achieve disinfection standard because of elevated MICs is debatable since significantly higher concentrations are used in practice (Russell and McDonnell 2000). 6. CONCLUSIONS The mechanisms of action of biocides are now better understood, although more research is needed in this field. It is generally accepted that most biocides, at high concentrations, act in a non-specific way. Random multiple detrimental effects that lead to cell death have been shown with highly reactive biocides and chemical agents used at high concentrations. When lower concentrations are used (i.e. inhibitory or sublethal concentrations) several studies have reported the emergence of resistant micro-organisms, although it is not clear at present whether those resistant micro-organisms are cross-resistant to other biocides and/or chemotherapeutic agents (Russell et al. 1999). Therefore, understanding the factors influencing biocidal activity

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

24S J . - Y . M A I L L A R D

remains a key issue. For example, the inability to appreciate the effects of dilution on the activity of a formulated product may lead to reduced efficacy in clinical applications and the emergence of resistant micro-organisms. The ultimate goal of studying the mechanisms of action of biocides should be (i) to improve the bactericidal activity of a product; (ii) to ensure that biocides are employed in the most advantageous manner and (iii) to prevent the emergence of resistant micro-organisms. Furthermore, understanding the target sites of a biocide will lead to improved antimicrobial formulations (Denyer and Stewart 1998). 7. REFERENCES Adams, E. (1968) Binding of Crystal Violet by Nucleic Acids of Escherichia coli. Birmingham: British Pharmaceutical Conference. Adams, R.L.P., Burdon, R.H., Campbell, A.M., Leader, D.P. and Smellie, R.M.S. (1981) The Biochemistry of Nucleic Acids, 9th edn. London: Chapman & Hall. Ayres, H.M., Furr, J.R. and Russell, A.D. (1999) Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa. Letters in Applied Microbiology 28, 13–16. Bellamy, W.R., Wakabayashi, H., Takase, M., Kawase, K., Shimamura, S. and Tomita, M. (1993) Role of cell-binding in the antibacterial mechanism of lactoferricin B. Journal of Applied Bacteriology 75, 478–484. Bloomfield, S.F. (1974) The effect of the phenolic antibacterial agent fentichlor on energy coupling in Staphylococcus aureus. Journal of Applied Bacteriology 37, 117–134. Bolle, A. and Kellenberger, E. (1958) The action of sodium lauryl sulphate on E. coli. Schweizerishe Zeitschrift fu¨r Pathologie und Bakteriologie 21, 714–740. Broadley, S.J., Jenkins, P.A., Furr, J.R. and Russell, A.D. (1995) Potentiation of the effects of chlorhexidine diacetate and cetylpyridinium chloride on mycobacteria by ethambutol. Journal of Medical Microbiology 43, 458–460. Brown, M.R.W. (1975) The role of the cell envelope in resistance. In Resistance of Pseudomonas aeruginosa pp. 71–99. London: John Wiley. Broxton, P., Woodcock, P.M. and Gilbert, P. (1983) A study of the antibacterial activity of some polyhexamethylene biguanides towards Escherichia coli ATCC 8739. Journal of Applied Bacteriology 54, 345– 353. Broxton, P., Woodcock, P.M. and Gilbert, P. (1984a) Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli. Journal of Applied Bacteriology 57, 115–124. Broxton, P., Woodcock, P.M. and Gilbert, P. (1984b) Injury and recovery of Escherichia coli ATCC 8739 from treatment with some polyhexamethylene biguanides. Microbios 40, 187–193. Cabral, J.P.S. (1991) Mode of antimicrobial action of dodine (dodecylguanidine monoacetate) in Pseudomonas syringae. Canadian Journal of Microbiology 38, 115–123. Chawner, J.A. and Gilbert, P. (1989a) A comparative study of the bactericidal and growth inhibitory activities of the bisbiguanides alexidine and chlorhexidine. Journal of Applied Bacteriology 66, 243– 252.

Chawner, J.A. and Gilbert, P. (1989b) Interaction of the bisbiguanides chlorhexidine and alexidine with phospholipid vesicles: evidence for separate modes of action. Journal of Applied Bacteriology 66, 253– 258. Chopra, I., Johnson, S.C. and Bennett, P.M. (1987) Inhibition of Providencia stuartii cell envelope enzymes by chlorhexidine. Journal of Antimicrobial Chemotherapy 19, 743–751. Ciak, J. and Hahn, F.E. (1967) Quinacrine (Atebrin): mode of action. Science 156, 655–656. Collier, P.J., Ramsey, A.J., Austin, P. and Gilbert, P. (1990) Growth inhibition and biocidal activity of some isothiazolone biocides. Journal of Applied Bacteriology 69, 569–577. Daltrey, D.L. and Hugo, W.B. (1974) Studies on the mode of action and the antibacterial agent chlorhexidine on Clostridium perfringens 2. Effect of chlorhexidine on metabolism and the cell membrane. Microbios 11, 131–146. Davies, A., Bentley, M. and Field, B.S. (1968) Comparison of the action of vantocil, cetrimide and chlorhexidine on Escherichia coli and its spheroplasts and the protoplasts of Gram-positive bacteria. Journal of Applied Bacteriology 31, 448–461. Davies, A. and Field, B.S. (1969) Action of biguanides, phenols and detergents on Escherichia coli and its spheroplasts. Journal of Applied Bacteriology 32, 233–243. Denyer, S.P. and Hugo, W.B. (1977) The mode of action of cetyltrimethylammonium bromide (CTAB) on Staphylococcus aureus. Journal of Pharmacy and Pharmacology 29, 66P. Denyer, S.P. and Stewart, G.S.A.B. (1998) Mechanism of action of disinfectants. International Biodeterioration and Biodegradation 41, 261–268. Dodd, C.E.R., Sharman, R.L., Bloomfield, S.F., Booth, I.R. and Stewart, G.S.A.B. (1997) Inimical processes: bacterial self-destruction and sub-lethal injury. Trends in Food Science and Technology 8, 238–241. Eklund, T. (1983) The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels. Journal of Applied Bacteriology 54, 383–389. Eklund, T. (1985a) The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli. Journal of General Microbiology 313, 73–76. Eklund, T. (1985b) Inhibition of microbial growth at different pH levels by benzoic and propionic acids and esters of p-hydroxybenzoic acid. International Journal of Food Microbiology 2, 159–167. El-Falaha, B.M.A., Furr, J.R. and Russell, A.D. (1989) Effect of anionic detergents on wild-type and envelope mutants of Escherichia coli and Pseudomonas aeruginosa. Letters in Applied Microbiology 8, 15–19. El-Falaha, B.M.A., Rogers, D.T., Russell, A.D. and Furr, J.R. (1985a) Effect of some antibacterial agents on the hydrophobicity of wild type and envelope mutant of Escherichia coli. Current Microbiology 12, 187–190. El-Falaha, B.M.A., Russell, A.D. and Furr, J.R. (1985b) Effects of chlorhexidine diacetate and benzalkonium chloride on the viability of wild type and envelope mutant of Escherichia coli and Pseudomonas aeruginosa. Letters in Applied Microbiology 1, 21–24. Ellison, R.T., Giehl, T.J. and La Force, F.M. (1988) Damage of the outer membrane of entire Gram-negative bacteria by lactoferrin and transferrin. Infection and Immunity 56, 2774–2781.

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

Favero, M.S. and Bond, W.W. (1991) Sterilization and antisepsis in the hospital. In Manual of Clinical Microbiology, 5th edn. ed. Balows, A., Hausler, W.J., Herrmann, K.L., Isenberg, H.D. and Shadomy, H.J. pp. 183–200. Washington: American Society for Microbiology. Fitzgerald, K.A., Davies, A. and Russell, A.D. (1989) Uptake of 14 C-chlorhexidine diacetate to Escherichia coli and Pseudomonas aeruginosa and its reversal by azolectin. FEMS Microbiology Letters 60, 327–332. Fitzgerald, K.A., Davies, A. and Russell, A.D. (1992) Effect of chlorhexidine and phenoxyethanol on cell surface hydrophobicity of Gram-positive and Gram-negative bacteria. Letters in Applied Microbiology 14, 91–95. Frederick, J.J., Corner, T.R. and Gerhardt, P. (1974) Antimicrobial action of hexachlorophene: inhibition of respiration in Bacillus megaterium. Antimicrobial Agents and Chemotherapy 6, 712–721. Gilbert, P., Beveridge, E.G. and Crone, B.P. (1977) The lethal action of 2-phenoxyethanol and its analogues upon Escherichia coli ATCC 5933. Microbios 19, 125–141. Gilbert, P., Pemberton, D. and Wilkinson, D.E. (1990) Barrier properties of the Gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides. Journal of Applied Bacteriology 69, 585–592. Gilby, A.R. and Few, A.V. (1960) Lysis of protoplasts of Micrococcus lysodeikticus by ionic detergents. Journal of General Microbiology 23, 19–26. Gittelson, B.L. and Walker, I.O. (1967) The interaction of proflavine with deoxyribonucleic acid and deoxyribonucleohistone. Biochemica and Biophysica Acta 138, 619–621. Gorman, S.P., Scott, E.M. and Russell, A.D. (1980) Antimicrobial activity, uses and mechanism of action of glutaraldehyde. Journal of Applied Bacteriology 48, 161–190. Greene, A.K., Few, B.K. and Serafini, J.C. (1993) A comparison of ozonation and chlorination for the disinfection of stainless steel surfaces. Journal of Dairy Science 76, 3612–3620. Haque, H. and Russell, A.D. (1974) Effect of ethylenediaminetetraacetic acid and related chelating agents on whole cell of Gramnegative bacteria. Antimicrobial Agents and Chemotherapy 5, 224–452. Heath, R.J., Yu, Y.-T., Shapiro, M.A., Olson, E. and Rock, C.O. (1998) Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. Journal of Biological Chemistry 273, 30316–30320. Hoffman, R.K. (1971) Toxic gases. In Inhibition and Destruction of the Microbial Cell ed. Hugo, W.B. pp. 225–258. London: Academic Press. Hughes, W.H. (1956) The Structure and Development of the Induced Long Forms of Bacteria ed. Spooner, E.T.C. and Stocker, B.A.D. Symposia of the Society for General Microbiology 6. Cambridge: Cambridge University Press. Hugo, W.B. (1971) Inhibition and Destruction of the Microbial Cell London: Academic Press. Hugo, W.B. (1978) Membrane-active antimicrobial compounds — a reappraisal of their mode of action in the light of the chemi-osmotic theory. International Journal of Pharmaceutics 1, 127–131. Hugo, W.B. (1999) Historical introduction. In Principles and Practice of Disinfection, Preservation and Sterilization, 3rd edn. ed. Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J. pp. 1–4. Oxford: Blackwell Scientific Publications.

25S

Hugo, W.B. and Bloomfield, S.F. (1971) Studies on the mode of action of the phenolic antibacterial agent Fentichlor against Staphylococcus aureus and Escherichia coli II. The effects of Fentichlor on the bacterial membrane and the cytoplasmic constituents of the cell. Journal of Applied Bacteriology 34, 569–578. Hugo, W.B. and Denyer, S.P. (1987) The concentration exponent of disinfectants and preservatives (biocides). In Preservatives in the Food, Pharmaceutical and Environmental Industries ed. Board, R.G., Allwood, M.C. and Banks, J.G. Society for Applied Microbiology Technical Series 22. pp. 281–291. Oxford: Blackwell Scientific Publications. Hugo, W.B. and Longworth, A.R. (1964) Effect of chlorhexidine diacetate on ‘protoplasts’ and spheroplasts of Escherichia coli, protoplasts of Bacillus megaterium and the Gram staining reaction of Staphylococcus aureus. Journal of Pharmacy and Pharmacology 16, 751–758. Hugo, W.B. and Longworth, A.R. (1965) Cytological aspects of the mode of action of chlorhexidine diacetate. Journal of Pharmacy and Pharmacology 17, 28–32. Hugo, W.B. and Longworth, A.R. (1966) The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic content, dehydrogenase activity and cell walls of Escherichia coli and Staphylococcus aureus. Journal of Pharmacy and Pharmacology 18, 569–578. Hugo, W.B. and Russell, A.D. (1999) Types of antimicrobial agents. In Principles and Practice of Disinfection, Preservation and Sterilization, 3rd edn. ed. Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J. pp. 5– 94. Oxford: Blackwell Scientific Publications. Ikeda, T., Tazuke, S. and Watanabe, M. (1983) Interaction of biologically active molecules with phospholipid membranes. I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain. Biochemica and Biophysica Acta 735, 380–386. Jones, D.S., Gorman, S.P., McCafferty, D.F. and Woolfson, A.D. (1991) The effects of three non-antibiotic antimicrobial agents on the surface hydrophobicity of certain micro-organisms evaluated by different methods. Journal of Applied Bacteriology 71, 218–227. Joswick, H.L., Corner, T.R., Silverdale, J.N. and Gerhardt, P. (1971) Antimicrobial actions of hexachlorophene: release of cytoplasmic materials. Journal of Bacteriology 108, 492–500. Kadohama, N. and McCarter, J.A. (1971) Inhibition of DNA polymerase of E. coli by proflavine. Canadian Journal of Biochemistry 50, 901–908. Kranz, R.G. and Lynch, D.L. (1979) Irgasan-induced pigmentation in Serratia marcescens and Pseudomonas aeruginosa. Microbios 24, 195– 207. Kroll, R.G. and Anagnostopoulos, G.D. (1981) Potassium leakage as a lethality index of phenol and the effect of solute and water activity. Journal of General Microbiology 50, 139–147. Kuyyakanond, T. and Quesnel, L.B. (1992) The mechanism of action of chlorhexidine. FEMS Microbiology Letters 100, 211–216. Lambert, P.A. and Hammond, S.M. (1973) Potassium fluxes. First indications of membrane damage in micro-organisms. Biochemical and Biophysical Research Communications 54, 796–799. Liau, S.Y., Read, D.C., Pugh, W.J., Furr, J.R. and Russell, A.D. (1997) Interaction of silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions. Letters in Applied Microbiology 25, 279–283.

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

26S J . - Y . M A I L L A R D

Maillard, J.-Y. and Russell, A.D. (2000) Biocides: activity and resistance — present status. In World Markets Series, Business Briefing: Global Health Care ed. Cooper, E. and Gahan, E. pp. 98–103. London: World Markets Research Centre. McDonnell, G. and Pretzer, D. (1998) Action and targets of triclosan. ASM News 64, 670–671. McMurry, L.M., McDermott, P.F. and Levy, S.B. (1999) Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan. Antimicrobial Agents and Chemotherapy 43, 711–713. McMurry, L.M., Oethinger, M. and Levy, S.B. (1998) Triclosan targets lipid synthesis. Nature, London 394, 531–532. Miall, S.H. and Walker, I.O. (1966) Structural studies on ribosomes I. The binding of proflavine to Escherichia coli ribosomes. Biochemica and Biophysica Acta 145, 82–95. Mitchell, P. (1961) Coupling of phosphorylation to electron and hydrogen transfer by chemiosmotic type of mechanism. Nature, London 191, 144–148. Mitchell, P. (1972) Chemiosmotic coupling in energy transduction: a logical development of biochemical knowledge. Journal of Bioenergetics 3, 5–24. Mlynarcik, D., Denyer, S.P. and Hugo, W.B. (1981) A study of the action of a bisquaternary ammonium salt, an amine oxide and an alkoxy phenylcarbamic acid ester on some metabolic functions in Staphylococcus aureus. Microbios 30, 27–35. Mudd, J.B., Reavitt, R., Ongun, A. and McManus, T.T. (1969) Reaction of ozone with amino acids and proteins. Atmospheric Environment 3, 669–681. Nakamura, K. and Tamaoki, T. (1968) Reversible dissociation of Escherichia coli ribosomes by hydrogen peroxide. Biochemica and Biophysica Acta 161, 368–376. Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial outermembrane permeability. Microbiology Review 49, 1–32. Olenick, J.G. and Hahn, F.E. (1972) Mode of action of primaquine: preferential inhibition of protein biosynthesis in Bacillus megaterium. Antimicrobial Agents and Chemotherapy 1, 259–262. Pearce, H., Messager, S. and Maillard, J.-Y. (1999) Effect of biocides commonly used in the hospital environment on the transfer of antibiotic-resistance genes in Staphylococcus aureus. Journal of Hospital Infection 43, 101–107. Prat, R., Nofre, C. and Cier, A. (1968) Effects de l’hypochlorite de sodium, de l’ozone et des radiations ionisantes sur les constituents pyrimidiques d’Escherichia coli. Annales de l’Institut Pasteur Paris 114, 595–607. Pulvertaft, R.J.V. and Lumb, G.D. (1948) Bacterial lysis and antiseptics. Journal of Hygiene, Cambridge 46, 62–64. Razin, S. and Argaman, M. (1963) Lysis of mycoplasma, bacterial protoplasts, spheroplasts and L-forms by various agents. Journal of General Microbiology 30, 155–172. Rego¨s, J. and Hitz, H.R. (1974) Investigations on the mode of action of triclosan, a broad spectrum antimicrobial agent. Zentralblatt fu¨r Bakteriologie und Hygiene, I. Abt Orig 226, 390–401. Russell, A.D. (1976) Inactivation of non-sporing bacteria by gases. In Inhibition and Inactivation of Vegetative Microbes ed. Skinner, F.A. and Hugo, W.B. Society for Applied Bacteriology Symposium Series No. 5. pp. 61–68. London: Academic Press. Russell, A.D. (1999) Factors influencing the activity of antimicrobial agents. In Principles and Practice of Disinfection, Preservation and

Sterilization, 3rd edn. ed. Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J. pp. 95–123. Oxford: Blackwell Scientific Publications. Russell, A.D. and Chopra, I. (1996) Antiseptics, disinfectants and preservatives: their properties, mechanisms of action and uptake into bacteria. In Understanding Antibacterial Action and Resistance, 2nd edn. pp. 97–149. London: Ellis Horwood. Russell, A.D. and Furr, J.R. (1977) The antimicrobial activity of a new chloroxylenol preparation containing ethylenediamine tetraacetic acid. Journal of Applied Bacteriology 43, 253–260. Russell, A.D., Furr, J.R. and Maillard, J.-Y. (1997) Microbial susceptibility and resistance to biocides: an understanding. ASM News 63, 481–487. Russell, A.D. and Hugo, W.B. (1994) Antimicrobial activity and action of silver. Progress in Medical Chemistry 31, 351–371. Russell, A.D. and McDonnell, G. (2000) Concentration: a major factor in studying biocidal action. Journal of Hospital Infection 44, 1–3. Russell, A.D., Suller, M.T.E. and Maillard, J.-Y. (1999) Do antiseptics and disinfectants select for antibiotic resistance? Journal of Medical Microbiology 48, 613–615. Salmond, C.V., Kroll, R.G. and Booth, I.R. (1984) The effect of food preservatives on pH homeostasis in Escherichia coli. Journal of General Microbiology 130, 2845–2850. Salton, M.R.J. (1968) Lytic agents, cell permeability and monolayer penetrability. Journal of General Physiology 52, 227S–252S. Salton, M.R.J. and Owen, P. (1976) Bacterial membrane structures. Annual Review of Microbiology 30, 451–482. Seiler, D.A.L. and Russell, N.J. (1991) Ethanol as food preservative. In Food Preservatives ed. Russell, N.J. and Gould, G.W. pp. 153–171. Glasgow: Blackie. Seligman, M.L. and Mandel, H.G. (1971) Inhibition of growth and RNA biosynthesis of Bacillus cereus by quinacrine. Journal of General Microbiology 68, 135–148. Serry, F.E.M., Denyer, S.P. and Hugo, W.B. (1986) Variations in E. coli cell morphology induced by 2-chloroacetamide. In Microbe 86, pp. G9–G65, Manchester: Proceedings, 14th International Congress of Microbiology. Silver, S.D. (1967) Acridine dye action at cellular and molecular levels. Experimental Chemotherapy 4, 505–511. Simon, E.W. (1953) Mechanism of dinitrophenol toxicity. Biological Review 28, 453–479. Simons, C., Walsh, S.E., Maillard, J.-Y. and Russell, A.D. (2000) A note: ortho-phthalaldehyde: proposed mechanism of action of a new antimicrobial agent. Letters in Applied Microbiology 31, 299–302. Singer, S.J. and Nicholson, G.L. (1972) The fluid mosaic model of the structure of cell membranes. Science 175, 720–731. Stewart, M.J., Parikh, S., Xiao, G., Tonge, P.J. and Kisher, C. (1999) Structural basis and mechanisms of enoyl reductase inhibition by triclosan. Journal of Molecular Biology 290, 859–865. Takasaki, A., Hashida, T., Fujiwara, S., Kato, K.-I. and Nishihara, T. (1994a) Bactericidal action of a quaternary ammonium disinfectant, didecyldimethyl ammonium chloride, against Staphylococcus aureus. Japanese Journal of Toxicology and Environmental Health 40, 344– 350. Takasaki, A., Hashida, T., Kato, K.-I., Moriyama, T. and Nishihara, T. (1994b) Action of a quaternary ammonium disinfectant on cell membrane of Staphylococcus aureus. Japanese Journal of Toxicology and Environmental Health 40, 520–526.

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S

ANTIBACTERIAL MECHANISMS OF ACTION OF BIOCIDES

Tattawasart, U., Maillard, J.-Y., Furr, J.R. and Russell, A.D. (1999) Comparative response of Pseudomonas stutzeri to antibacterial agents. Journal of Applied Microbiology 87, 323–331. Thurman, R.B. and Gerba, C.P. (1989) The molecular mechanisms of copper and silver ion disinfection of bacteria and viruses. Critical Reviews of Environmental Control 18, 295–315. Trias, J. and Benz, J. (1994) Permeability of the cell wall of Mycobacterium smegmatis. Molecular Microbiology 14, 283–290. Turner, E.J. (1983) Hydrogen peroxide and other oxidant disinfectants. In Disinfection, Sterilization and Preservation, 3rd edn. ed. Block, S.S. pp. 240–250. Philadelphia: Lea and Febiger. Ulitzur, S. (1970) The transport of b-galactosides across the membrane of permeaseless Escherichia coli ML35 cells after treatment with cetyltrimethylammonium bromide. Biochemica and Biophysica Acta 211, 533–541.

27S

Vaara, M. (1992) Agents that increase the permeability of the outer membrane. Microbiology Reviews 56, 395–411. Vaara, M. and Vaara, T. (1983a) Polycations sensitise enteric bacteria to antibiotics. Antimicrobial Agents and Chemotherapy 24, 107–113. Vaara, M. and Vaara, T. (1983b) Polycations as outer membranedisorganizing agents. Antimicrobial Agents and Chemotherapy 24, 114–122. Voss, J.G. (1963) Effect of inorganic cations on bactericidal activity of anionic surfactants. Journal of Bacteriology 86, 207–211. Walsh, S.E., Maillard, J.-Y., Simons, C. and Russell, A.D. (1999) Studies on the mechanisms of the antibacterial action of orthophthalaldehyde. Journal of Applied Microbiology 87, 702–710. Wang, J.H. and Matheson, A.T. (1967) The possible role of sulphydryl groups in the dimerization of 70S ribosomes from Escherichia coli. Journal of Biological Chemistry 23, 740–744.

Ó 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 16S–27S