International Biodeterioration 26 (1990) 89-100

Mechanisms of Action of Biocides

S. P. De n y e r
Department of Pharmaceutical Sciences, University of Nottingham,
University Park, Nottingham NG7 2RD, UK
ABSTRACT
Detailed studies have clearly demonstrated that f ew biocides can be
considered now as general cell poisons. Biocidal action may result through
physicochemical interaction with microbial target structures, specific reac-
tions with biological molecules, or disturbance of selected metabolic or
energetic processes. Mechanism of action studies, i f intelligently applied, can
provide direction to the development of novel biocides and biocidal
systems.
I NT RODUCT I ON
Bi oci des c ompr i s e a he t e r oge ne ous gr oup of c he mi c a l agent s, of t en well
char act er i s ed i n t hei r be ha vi our i n di verse appl i cat i ons but f r equent l y
little u n d e r s t o o d i n t er ms of t he basi s for t hei r activity. Ther e is t hus a
gr owi ng ne e d to est abl i sh me c h a n i s ms of act i on for bi oci des to assi st in
t he des i gn of ne w c o mp o u n d s or c o mb i n a t i o n s of c o mp o u n d s , i n t he
u n d e r s t a n d i n g of r esi st ance me c ha ni s ms , a nd to pr ovi de a focus for
t oxi col ogi cal at t ent i on. Cer t ai nl y, me c h a n i s m of act i on st udi es have
s hown t hat bi oci dal agent s can no l onger be c ons i de r e d as gener al cell
poi s ons a n d t her ef or e t hei r act i vi t y ma y be opt i mi s e d by desi gn. The
obj ect of t hi s s hor t review, is to i l l ust r at e ma j or me c h a n i s ms of act i on
wi t h sel ect ed exampl es chos en f r om t he ant i bact eri al literature. Al t hough
not al ways di r ect l y appl i cabl e, ma n y of t he pr i nci pl es cover ed will al so
a ppl y to act i on agai ns t eukar yot i c cells.
89
International Biodeterioration 0265-3036/90/$03.50 -- 1990 Elsevier Science Publishers
Ltd, England. Printed in Great Britain.
90 S. P Denyer
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
Structural damage
Leakage
Autolysis
Bacteriostasis
Inability to repair
Lysis
Cytoplasm coagulation Bactericidal
Fig. 1. The consequence of biocide-induced damage.
Mechanisms of action of biocides 91
a . b . c . d . e .
J
EQUiLiBRIUM
f
CONCENTRATION
/
#
Fig. 2. Types of sorpt i on i sot herm (as pr oposed by Gi l es et al. (1960, 1974a, b), Giles &
McKay (1965) and Giles & Toila (1964)): (a) S-shape; (b) L-shape: (c) H- shape: (d) C-
shape: (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 conveni-
ently 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 sub-
strate 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.
92 S. P. Denyer
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Mechanisms of action of biocides 93
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94 S. P. Denyer
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Fig. 3. Biocide interactions with the cytoplasmic membrane: (a) Insertion of positively-
charged biocide into phosphol i pi d bilayer and attraction of negatively-charged
phosphol i pi ds e.g. CTAB; (b) reorganisation of phosphol i pi d distribution through
surface interaction with biocide possessing multiple positively-charged centres e.g.
pol ymeri c biguanides; (c) di spl acement of phosphol i pi ds and disorganisation of
membr ane architecture e.g, phenols; (d) pore format i on through biocide self-association
e.g. low concentrations of sodium dodecyl sulphate; (e) solubilisation of membr ane-
bound protein (and phosphol i pi d) e.g. high concentrations of anionic detergents.
Mechanisms of action of biocides 95
TABLE 2
Some Synergistic Combi nat i ons of Biocides Where a Mechani sm of Synergy Has Been
Proposed (modified from Denyer & King, 1988)
Synergistic combinations Proposed mechanism Reference
Phenyl mercuri c acetate and 3-cresol
or benzal koni um chloride
Lipophilic weak acids and fatty
alcohols
Chl orhexi di ne and Bronopol
Acetate and lipophilic weak acids
N-chloramines and diazolidinyl urea
(Germal l 2)
Chlorocresol and 2-phenylethanol
Permeabilisation
Permeabilisation
and bi ochemi cal
enhancement
Permeabilisation
Biochemical
enhancement
Permeabilisation
and bi ochemi cal
enhancement
Biochemical
enhancement
Hugbo (1977)
Comer (1981)
Woznia k-Pamowska
& Krowczynski
(1981)
Moon (1983)
Llabres & Aheam
(1985)
Denyer et al. (1986)
APPLI CATI ON OF MECHANI SMS OF ACTION TO
BI OCI DE DESI GN
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 underst andi ng the biochemical basis of action, it is also possible to
identify potentially synergistic combi nat i ons of biocides, i. e. those
offering enhanced activity upon combi nat i on (Table 2). Here, the mutual
interrelationship between bi ochemi cal and biophysical functions of the
cell is exploited by selecting biocides that can mi ni mi se cellular recovery
and maximise breadt h of damage (biochemical synergy). Further, some
biocides can increase cellular permeability and can therefore be used to
advantage in enhanci ng the action of intracellularly-active agents
(permeabilisation synergy). Ext endi ng this latter concept, it is possible to
couple intracellularly-acting biocidal agents to actively transported
substrates in order to exploit the nat ural concentrating potential of the
cell (portage transport). Int racel l ul ar cleavage of biocide from substrate
will t hen permit biocidal activity. Such an approach offers potential
selective toxicity advantages to biocide systems (Table 3).
9 6 S. P. Denyer
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Mechanisms of action of biocides 97
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