Mechanisms of action of biocides

International Biodeterioration 26 (1990) 89-100

Mechanisms of Action of Biocides

S. P. D e n y e r Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK

ABSTRACT Detailed studies have clearly demonstrated that few biocides can be considered now as general cell poisons. Biocidal action may result through physicochemical interaction with microbial target structures, specific reactions with biological molecules, or disturbance of selected metabolic or energetic processes. Mechanism of action studies, if intelligently applied, can provide direction to the development of novel biocides and biocidal systems.

INTRODUCTION Biocides comprise a heterogeneous group of chemical agents, often well characterised in their b e h a v i o u r in diverse applications but frequently little u n d e r s t o o d in terms of the basis for their activity. There is thus a growing need to establish m e c h a n i s m s of action for biocides to assist in the design of new c o m p o u n d s or c o m b i n a t i o n s of c o m p o u n d s , in the u n d e r s t a n d i n g of resistance m e c h a n i s m s , a n d to provide a focus for toxicological attention. Certainly, m e c h a n i s m of action studies have shown that biocidal agents can no longer be considered as general cell poisons a n d therefore their activity may be optimised by design. The object of this short review, is to illustrate major m e c h a n i s m s of action with selected examples chosen from the antibacterial literature. Although not always directly applicable, m a n y of the principles covered will also apply to action against eukaryotic cells. 89 International Biodeterioration 0265-3036/90/$03.50 -- © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain.

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CONSEQUENCES OF BIOCIDE INTERACTION Antibacterial agents may exert both bacteriostatic and bactericidal effects, often in a concentration-dependent manner. The mechanisms of action responsible for each effect may not necessarily be the same. Bacteriostatic events can be considered generally to result from some form of metabolic inhibition which is released upon removal of the biocide, while bactericidal action is caused by irreversible or irreparable damage to a vital structure or function of the cell (Fig. 1). In many instances, damage arising from interactions may theoretically be repairable, but unless appropriate repair processes can be successfully initiated at an early stage and the damaging event be halted then cell death will occur. Thus it is essential that in the study of mechanisms of action attention is paid to correct and effective methods for cell recovery and biocide inactivation.

STAGES OF INTERACTION All biocides, irrespective of their final effect, undergo a number of stages of interaction embracing uptake, partition to target(s), and concentration at target(s), before finally eliciting the damaging event(s). The extent and kinetics of uptake are characterised by the sorption isotherm (Fig. 2) which identifies high affinity and Langmuir uptake (H and L, respectively; e.g. CTAB, chlorhexidine, dequadin), constant uptake and co-operative sorption (C and S, respectively; e.g. some phenols) and enhanced uptake (Z; e.g. 2-phenoxyethanol; Gilbert et al. (1978)). Consequent upon, and deriving from, biocide uptake is the phase of partition which differs in rapidity and extent depending upon the Selective permeability changes (uncoupling, transport inhibition) Interaction with nucleic acids Enzyme inhibition

Bacteriostasis

Structural damage Leakage Autolysis

Inability to repair

Lysis Cytoplasm coagulation

Bactericidal

Fig. 1. The consequence of biocide-induced damage.

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Fig. 2. Types of sorption isotherm (as proposed by Giles et al. (1960, 1974a, b), Giles & McKay (1965) and Giles & Toila (1964)): (a) S-shape; (b) L-shape: (c) H-shape: (d) Cshape: (e) Z-shape.

physicochemical characteristics of the biocide, and nature of cellular barriers, and the extent of non-specific interactions arising with cellular components (Gilbert & Wright, 1987). The ability of the biocide to concentrate at specific sites within the bacterium then determine the potential sensitivity of individual targets to that agent and the eventual damaging event(s).

POTENTIAL TARGETS AND TYPES OF DAMAGE Target regions for antibacterial agents can be classified very conveniently as the cell wall, cytoplasmic membrane and cytoplasm. Within these broad areas of the cell a further division of targets can be made into those of biochemical or structural significance. These divisions are created for convenience only and do not represent mutually exclusive areas for biocide interaction. Indeed, many of the biocides currently in use have more than one potential target within the bacterial cell (Table 1), and the strong interdependence of cellular functions cannot be ignored. Undoubtedly, the focus for many agents is the cytoplasmic membrane, interactions at this level frequently causing fundamental changes in both membrane structure (Fig. 3) and function. This has important implications for the entire cell biochemistry with disturbance of respiration, cellular energetics, transport processes, intracellular substrate reservoirs, and enzyme function arising. In such an event, and with such widespread resultant damage, care must be taken not to overlook the prime lesion.

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TABLE 2

Some Synergistic Combinations of Biocides Where a Mechanism of Synergy Has Been Proposed (modified from Denyer & King, 1988)

Synergistic combinations

Proposed mechanism

Reference

Phenylmercuric acetate and 3-cresol or benzalkonium chloride Lipophilic weak acids and fatty alcohols

Permeabilisation

Hugbo (1977)

Permeabilisation and biochemical enhancement Permeabilisation

Comer (1981)

Chlorhexidine and Bronopol

Acetate and lipophilic weak acids N-chloramines and diazolidinyl urea (Germall 2) Chlorocresol and 2-phenylethanol

Biochemical enhancement Permeabilisation and biochemical enhancement Biochemical enhancement

Woznia k-Pamowska & Krowczynski (1981) Moon (1983) Llabres & Aheam (1985) Denyer et al. (1986)

APPLICATION OF MECHANISMS OF ACTION TO BIOCIDE DESIGN Recognition of the specific target for a biocide allows determination of those features necessary to enable that agent to reach, and interact with, that target. Through this knowledge, quantitative structure-activity relationships may be sought in groups of related compounds in order to optimise activity. By understanding the biochemical basis of action, it is also possible to identify potentially synergistic combinations of biocides, i.e. those offering enhanced activity upon combination (Table 2). Here, the mutual interrelationship between biochemical and biophysical functions of the cell is exploited by selecting biocides that can minimise cellular recovery and maximise breadth of damage (biochemical synergy). Further, some biocides can increase cellular permeability and can therefore be used to advantage in enhancing the action of intracellularly-active agents (permeabilisation synergy). Extending this latter concept, it is possible to couple intracellularly-acting biocidal agents to actively transported substrates in order to exploit the natural concentrating potential of the cell (portage transport). Intracellular cleavage of biocide from substrate will then permit biocidal activity. Such an approach offers potential selective toxicity advantages to biocide systems (Table 3).

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