INTERNATIONAL

BlODETERIORATION&
BIODEGRADATION
International Biodeterioration & Biodegradation 41 (1998) 261-268

Mechanisms of action of disinfectants

S. P. Denyera,* G. S. A. B. Stewartb
Department qf Pharmacy, University of Brighton. Lewes Road, Brighton, BN2 4GJ, UK
bDepartment of Pharmaceutical Sciences. University of Nottingham. Unitersit}~ Park, Nottingham. NG7 2RD. UK

Accepted; 29 January 1998

Abstract

Disinfectants and biocides are a chemically diverse group of agents which are generally considered to exhibit poor selective toxicity.
This should not be mistaken for poor target specificity, however, and much is now known concerning the damaging interactions
which may arise between bacterial cell and disinfectant agent. Critical governing features of these interactions are the physicochemical
characteristics of the chemical agent, cell morphology, and the physiological status of the microorganism. Antibacterial events
include membrane disruption, macromolecule dysfunction, and metabolic inhibition; the consequential effect is determined by the
relative contribution(s) of the target(s) to microbial cell survival and the possible initiation of self-destructive processes. Disinfection
kinetics offer a measure to differentiate between physiochemical and chemical interactions.
Increasingly demanding disinfectant applications require more sophisticated use of biocidal systems. Approaches include: agents
in combination, whereby a knowledge of mechanism of action assists in designing optimal mixtures; intracellular biocide delivery,
using cellular transport processes to overcome cellular barriers; and targeted donation of biocide from delivery systems, requiring
an understanding of target reactivity.
A knowledge of disinfection mechanisms provides a basis from which novel chemistries and synergistic combinations may be
developed. 0 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Disinfectants; Mode of action; Cellular targets; Antibacterial activity.

1. Introduction to present a view on possible future developments; the

methods employed to establish these lesions are discussed
Disinfectants, or chemical biocides to use a more general in an earlier treatment by Denyer and Hugo (1991a).
term, differ from antibiotic agents in their relative lack More detailed examination of individual agents can be
of selective toxicity and an often implied lack of target found in the reviews and monographs of Hugo (1991,
specificity. The former characteristic leads to an ever- 1992, 1995); Hugo and Russell (1992); Paulus (1991,
increasing pressure to reduce concentrations used and 1993). A broader perspective has recently been published
maximise efficacy, while the latter assumption dissuades by Russell et al. (1997).
against rational design and encourages empirical solu-
tions to the former problem. A careful examination of
the literature however, discloses that the basis of biocide 2. Early stages of cell-biocide interaction
action offers a fertile area for scientific exploration with
the potential to establish principles by which improved The vegetative bacterial cell offers three broad regions
agents can be developed. Thus, mechanism of action for biocide interaction: the cell wall, cytoplasmic mem-
studies can provide direction to novel chemistries and brane and cytoplasm (Fig. 1). Biocide access to these
delivery systems, offer predictive capability for com- regions is determined by extracellular material, cell mor-
bination biocides, may give clues to the basis of unwanted phology and cellular chemical composition; phenotypic
toxicity. and can help elaborate the often complex variation in cell physiology may lead to intrinsic resist-
homeostatic processes associated with cell survival. ance (Nicas and Hancock, 1980; Broxton et al., 1984;
This review seeks to summarise the principal anti- Wright and Gilbert, 1987; Brown et al., 1990).
bacterial lesions associated with chemical biocides and The initial interaction with the bacterial cell is
described by the uptake isotherm (Denyer, 1990); effec-
tive biocide action requires subsequent partitioning of
* Corresponding author. the sorbed biocide to the target site followed by accumu-

SO96&8305/98/$19.00 xo 1998 Elsevier Science Ltd. All rights reserved.

PII: SO964-8305(98)00023-7
262 S.P. Denyer and G.S.A.B. Stewart/International Biodeterioration & Biodegradation 41 (1998) 261-268
Gram negative
(outer membrane)
Fig. 1. Potential targets for biocides (reproduced with permission from Denyer, 1995).
lation to damaging levels, characteristics strongly influ-
enced by biocide physicochemistry (Denyer, 1995). In
progression to their targets, biocides encounter a variety
of structures; experience shows that the lipidic outer
membrane of the Gram-negative bacterium is dominant
among these. Hydrophilic biocides of molecular weight
less than 600 Da are very likely to pass through the water-
filled porins; for other agents of greater size or lipo-
philicity this structure is likely to offer a quite con-
siderable hindrance to passage (Russell, 1991; Gilbert
and Wright, 1987). Frequently, penetration must then
be assisted by localised destabilising effects in the outer
membrane arising from damaging interactions (eg.
EDTA; Russell, 1991), by biocide molecular co-associ-
ation altering the physicochemistry of partition (Gilbert
and Al-Taae, 1985; Kanazawa et al., 1995) or through
formulation design. Initial sorption and progression to,
and accumulation at, target are rarely instantaneous
events and can therefore be influenced by factors such as
biocide concentration, temperature and formulation; pH
will affect both the charge on microbial structures and
the ionization of ionizable biocides, again affecting the
initial interactive stages (Richards et al., 1995). Thus,
extraneous factors may profoundly influence the rate and
extent of cell injury without necessarily invoking any
direct effect on the mechanism of action.
3. Potential targets for biocides
From the perspective of functionality the cell wall,
comprising an open network of peptidoglycan (with
1 Gram positive
Cytoplasmic membrane
structural integrity
@Respiratory chain and
membrane-bound enzymes
6 Transport mechanisms
0 Energy coupling processes
Cell wall peptidoglycan
- Cytoplasmic membrane
l Nucleic acids
2
a lipopolysaccharide overlayer for Gram-negative
bacteria), provides for little more than structural integ-
rity; in this regard, it serves as an excellent target for
antibiotics but rarely proves a suitable site for direct
biocide action. The cytoplasmic membrane however
offers a rich matrix of balanced interactions between pho-
spholipid and enzymic/structural protein, ensuring a con-
trolled impermeability and topological organisation by
which intracellular homeostasis and vectorial trans-
port/metabolism are maintained (Singer and Nicolson,
1972; Salton and Owen, 1976; Kroll and Patchett, 199 1);
this multiplicity of critically sensitive functions and the
large expanse for interaction predispose this region to
biocide attack. Many catabolic and anabolic processes
are vested in the cytoplasm, together with the necessary
replicative machinery; this region also affords a diverse
range of target processes but, by virtue of its inclusion
within the cytoplasmic membrane, it is the last region for
biocide accumulation. Thus, the target most frequently
cited in the biocide literature is the cytoplasmic
membrane, particularly in investigations of bacteriostasis
where biocide concentrations are relatively low and the
membrane appears especially sensitive to functional per-
turbation (Fig. 1). At disinfection levels however, it may
be more difficult to distinguish between lesions occurring
at the membrane and the cytoplasm.
Biocide physicochemistry will affect the efficacy of tar-
get interaction. For electrophilic agents, which include
the aldehydes, active-halogen compounds, isothiazo-
linones and the N-haloalkylthio biocides, reactivity is
dependant upon low steric hindrance and strong electron-
 S.P. Denyer and G.S.A.B. Stewart/International Biodeterioration & Biodegradation 41 (1998) 261-268 263

withdrawing capability. Membrane perturbing agents are Anagnostopoulos, 198 1; Daham and Wiseman, 1987;
governed in their interactions by the balance between Hugo and Denyer, 1987).
hydrophobic and polar groups; quantitative structure Biocidal compounds derive from a variety of chemical
activity relationships equate partition coefficients with classes; the precise mechanisms of interaction often reflect
the activity of phenols, weak acids and their esters, while this diversity although the final damaging outcomes may
the positively charged head group and alkyl chain length show considerable similarity (Table 2). Damage may be
determine the relative efficiencies of cationic surface- manifest in the following ways:
active agents. A close examination of biocide-target inter-
actions indicates a differentiation between strong chemi- l disruption of the transmembrane proton motive force
cal (covalent) or ionic interactions and those associated leading to an uncoupling of oxidative phosphorylation
with physical partitioning. The former interactions are and inhibition of active transport across the mem-
characterised by a general tolerance to concentration brane;
l inhibition of respiration or catabolic/anabolic reac-
change often requiring the use of specific neutralizers
for inactivation, while those driven by partitioning are tions;
l disruption of replication;
readily inactivated by dilution. This behaviour is reflected
l loss of membrane integrity resulting in leakage of
in the concentration exponent (Table 1); knowledge of
this parameter and its determination in disinfection kin- essential intracellular constituents such as potassium
etics (Reichart, 1994) may allow presumptive conclusions cation, inorganic phosphate, pentoses, nucleotides and
to be drawn regarding the mechanisms of inter- nucleosides, and proteins;
action/action (Hugo and Denyer, 1987). 0 lysis;
0 coagulation of intracellular material.

4. Mechanisms of antibacterial action
The classical approach to determining a mechanism
of action relies upon establishing a correlation between
concentrations initiating bacteriostatic or bactericidal
effects and those precipitating specific biochemical or
physiological changes (Bloomfield, 199 1); where cor-
relation is seen a causal relationship is implied. As might
be expected, experimental conditions may profoundly
affect the concentration threshold of whole cell events
and considerable care must be taken to properly account
for experimental artefacts (Russell et al., 1973; Denyer
and Hugo, 1991a). An alternative approach may be to
explore the relationship of severity of effect at the cellular
and biochemical levels with some intrinsic property of
the biocide; concentration exponent, sorption profiles,
rate kinetics and temperature coefficients have all been
used for this purpose (Gilbert et al., 1977; Kroll and
Taken in order, these lesions represent injury of increas-
ing severity spanning bacteriostasis to rapid bactericidal
action. Conventional theory recognises progressive injury
arising from initial lesions and if the inimical challenge is
maintained for sufficient duration or the applied con-
centration is sufficiently high then initial bacteriostatic
damage may develop to cause eventual cell death.
Evidence is now accumulating to suggest that the tran-
sition from sub-lethal injury to cell death may also arise
following the initiation by biocide of self-destructive
events. Metabolic imbalance following biocidal and other
stresses has been postulated to lead to free radical pro-
duction and self-destruction, a phenomenon which read-
ily differentiates highly metabolically active exponentially
growing cells from those in stationary phase (Dodd et al.,
1997). Evidence for free radical action is provided by
inducible ox,~R protective effects (Greenberg and
Demple, 1986; Chapman and Diehl, 1995) catalase-

Table 1
Characteristics of interaction for selected biocides

Concentration Inactivating/neutralizing
Example biocides exponent Site of of action Type of interaction process

Bronopol <2 Enzyme-SH Chemical Cysteine

QACs Membrane Ionic Lecithin + Tween 80
Formaldehyde Amino groups Chemical Glycine
Biguanides Membrane Ionic Lecithin + Tween 80
Sorbic acid 224 Enzyme-SH Chemical Dilution + Tween 80
Proton gradient Physical
Ethanol >4 Membrane Physical Dilution
Phenols Membrane Physical Dilution
Aromatic alcohols Membrane Physical Dilution
264 S.P. Denver and G.S.A.B. Stewart/International Biodeterioration & Biodegradation 41 (1998) 261-268

Table 2
Mechanisms of interaction of selected biocides with their prospective targets and the consequential antibacterial effects

Mechanisms of interaction Example biocides Typical targets Antibacterial effect

Chemical reactions:
Oxidation of (predominantly) Isothiazolinones, Thiol-containing cytoplasmic and Metabolic inhibition
thiol groups organomercurials, heavy metal membrane-bound enzymes e.g.
salts. hypochlorites, dehydrogenases
organochlorine derivatives,
bronopol. metallo-compounds
(e.g. oxines)

General alkylation reactions Glutaraldehyde, formaldehyde Biomolecules (e.g. protein, RNA, Metabolic and replicative
(and by implication DNA) containing amino, imino, inhibition; possibly some cell wall
formaldehyde-releasing agents), amide, carboxyl and thiol groups damage
chloroacetamide (nucleophiles). Intermolecular
cross-linking may occur
Halogenation Hypochlorites, chlorine-releasing Amino groups in proteins Metabolic inhibition
agents

Free-radical oxidation (e.g. Hydrogen peroxide, peracetic Enzyme and protein thiol groups Metabolic inhibition
hydroxyl radicals) acid

Metal ion chelation EDTA, oxines Divalent cation-mediated outer Leakage; increased susceptibility
membrane integrity, principal to applied stress. Metabolic
target region Gram-negative cell inhibition
wall (EDTA). Metal ion-requiring
enzyme processes
Intercalation Aminoacridines Intercalation between DNA base Replicative injury
pairs
Ionic interactions:
Electrostatic (ionic) interaction QACs, chlorhexidine, Cytoplasmic membrane integrity; Leakage; respiratory inhibition;
with phospholipids polyhexamethylene biguanides membrane-bound enzyme intracellular coagulation
environment and function
Physical interactions:
Penetration/partition into Phenols, weak acids, parabens. Transmembrane pH gradient; Leakage; disruption of transport,
phospholipid bilayer; possible tetrachlorosalicylanilide, membrane integrity respiratory and energy coupling
displacement of phospholipid phenoxyethanot fentichlor, 2- processes
molecules; intramembrane phenylethanol
molecular cycling

Solution of phospholipids Aliphatic alcohols Membrane integrity Leakage

Membrane-protein solubilization Anionic surfactants Cytoplasmic membrane integrity; Leakage, uncoupling of energy
membrane-bound enzyme processes; lysis
environment and function

enhanced survival (Martin et al., 1976; Flowers et al., metabolic imbalance and impaired ionic homeostasis
1977; van Netten et al., 1984) and improved viable cell (Table 3). It might be postulated that many of the inhibi-
recovery in minimal media (Tang and Jackson, 1979; tors of respiration and oxidative phosphorylation (Di
Gilbert, 1984). A mechanism of action involving free
radical generation has been proposed for the biocides 2-
bromo-2-nitropropane-1,3-diol (Bronopol; Stretton and
Table 3
Manson, 1973; Shepherd et al., 1988) and 5-chloro-2-
Examples of autocidal effects
methyl-isothiazolin-3-one (Chapman and Diehl, 1995).
Thus the effect of a biocide may be supplemented by Autocidal effect Initiating processes
autocidal activity (Fig. 2, biocide 1); once initiated this
process may account for a significant proportion of popu- Free-radical accumulation through Anabolic suppression
metabolic imbalance Enhanced catabolism
lation death and may not be resolvable by conventional
Respiratory stimulation
biocide neutralizers (Table 1). Mechanisms of action Redox chain perturbation
which may lead to such autocidal processes currently fall Activation of latent ribonucleases Impaired ionic homeostasis
into two categories: free radical accumulation through
 S.P. Denyer and G.S.A.B. Stewart/International Biodeterioration & Biodegradation 41 (1998) 261-268
Reversible
bacteriostatic
I effect
APPLIED BIOCIDE STRESS
Fig. 2. Nature of the antibacterial effect arising from treatment
concentration or contact time.
Monte et al., 1984; Kroll and Patchett, 1991; Phillips-
Jones and Rhodes-Roberts, 1991) might lie in the former
category, while membrane-active agents precipitating
potassium ion leakage and thereby affecting intracellular
homeostasis may initiate autolysis through activation of
latent ribonucleases (Denyer and Hugo, 1991b; Lambert
& Smith 1976). Autocidal effect may be prevented or
limited, for example by the addition of free radical detox-
ifiers or by low temperature inhibition of autolytic
enzymes (Denyer and Hugo, 1991b), but presumably
early action would be required to have any significant
effect on survival. Biocides which have no effect on meta-
bolic activity or cellular homeostasis are unlikely to pre-
cipitate an autocidal process (Fig. 2, biocide 2); there is
thus good reason to explore mechanisms of action in a
search for this advantageous compounding effect.
5. Strategies to enhance mechanism of action
With a knowledge of interaction chemistry and anti-
bacterial lesions, strategies begin to emerge by which
biocidal action may be improved. These may include
approaches to enhance initial interaction or accumu-
265
l Topological destruction
l Enzyme denaturation
l Cytoplasm coagulation
I 1
I I
I I
1 I
I 1
i Preventable i Irreversible
I autocidal i bactericidal
I I
I effect I effect
->
with two classes of biocide, 1 and 2. Applied biocide stress may be represented by
lation at target site, optimisation of synergistic biocidal
combinations, or engaging the bacterium in a part-
nership with the biocide to invoke autocidal processes.
5.1. Enhanced interaction
Leeming et al. (1997) have described the physisorption
of isothiazolinone analogues to hydrophobic beads with
an optimised hydrophile-lipophile balance to ensure
direct donation to an approaching bacterium (Fig. 3).
This permits the use of biocides with low water solubility
but optimal partition characteristics for the Gram-nega-
tive cell envelope. Preliminary studies indicate a bac-
tericidal effect in excess of four decimal reductions against
Pseudomonas aeruginosa.
5.2. Intracellular biocide delivery
Substrate carriers may be employed to facilitate the
intracellular accumulation of membrane-impermeable
biocides. By employing Escherichia coli genetically engi-
neered to bioluminesce, Denyer et al. (199 1) have dem-
onstrated the intracellular delivery of a range of novel
266 S.P. Denyer and G.S.A.B. Stewart/International Biodeterioration & Biodegradation 41 (1998) 261-268

l Presentation of biocide on
demand from an immobilized
support

l Permits optimal hydrophilic-

lipophilic balance aiding
biocide uptake and
penetration to target

Fig. 3. Biocide donation from an immobilized support (modified from Leeming et al., 1997).

phenolic derivatives of galactose via the lac permease. synergistic potential; a knowledge of the interactions
Intracellular cleavage of biocide from the transported involved will guide formulation design.
molecule resulted in cell damage and loss of metabolic
function as indicated by reduced light output (Fig. 4). 5.4. Autocidal effect

5.3. Optimised biophysicallbiochemical synergy The identification of an autocidal component to bioc-

The literature reports a variety of synergistic com-
binations (for example, Lehmann (1988)); a close exam-
ination would suggest that a selection offer readily
identifiable mutually supportive mechanisms of action
(Denyer, 1996). Denyer et al. (1986) have demonstrated
that the mechanism of synergy in biocide combinations
can be investigated experimentally. Such a complex mul-
tivariate data set requires statistical interpretation
(Rabone and Harbron, personal communication) aided
by simple factorial design for assessment of activity (Kar-
abit et al., 1989). Close examination of mechanisms of
action may thus offer a route to predicting and identifying
ide action offers an exciting challenge to the disinfectant
formulator. Metabolic imbalance arising from an
uncoupling of anabolic and catabolic processes will be
catalysed at sub-lethal biocide levels. Thus, this phenom-
enon may become of greatest import in circumstances
where biocide accumulation at target is limited and the
cell is still metabolically operational. It is therefore poss-
ible to conceive of situations where controlled, possibly
sequential, additions of biocides might initiate autocidal
processes more efficiently than a high initial dose. What
relevance also the autocidal process to the biofilm organ-
ism, established to survive in a nutrient deplete environ-
ment? Could we here present challenges to conventional

120

80
Bioluminescence
after 24h 60
incubation 4c)
as % of control
20

PC; PMPG PBPG TBPG DCPG PCONPG EPHBG

Biocide Derivative
Fig. 4. Bioluminescence from Escherichia coli pSBlO0 growing in the presence of selected P-D-galactopyranoside (G) derivatives of phenolic biocides.
PG, phenyl-G; PMPG, 4-methylphenyl-G; PBPG, 4-bromophenyl-G; TBPG, (3,4,5)-tribromophenyl-C; DCPG, (2,4)-dichlorophenyl-G; PCONPG.
4-chloro-2-nitrophenyl-G; EPHBG, ethyl 4-hydroxybenzoate-G.
 S.P. Denyer and G.S.A.B. Sfewart/International Biodeterioration & Biodegradation 41 (1998) X-268 267

wisdom: might deliberate metabolic activation of con- G.S.A.B., 1997. Inimical processes: bacterial self-destruction and
sub-lethal injury. Trends Food Sci. Technol. 8, 238-241.
taminating organisms predispose to intracellular injury?
Flowers, R.S., Martin, S.E., Brewer, D.G.. Ordal, Z.J., 1977. Catalase
and enumeration of stressed Staph.vlococcus aweus cells. Appl. Env.
Microbial. 33, 1112-I 117.
6. Conclusion Gilbert. P.. 1984, The revival of micro-organisms sublethally injured
by chemical inhibitors. In: The Revival of Injured Microbes, ed.
The study of biocide mechanisms of action offers an, M.H.E. Andrew and A.D. Russell. pp. 175-197. Academic Press,
London.
as yet, largely untapped initiator of novel development
Gilbert, P., Al-Taae, A., 1985. Antimicrobial activity of some alkyl-
directions. Quantitative structure-activity relationships trimethylammonium bromides. Lett. Appl. Microbial. 1, 101-104.
will provide unique insight into fruitful chemical per- Gilbert. P., Wright, N.E.. 1987. Non-plasmidic resistance towards pres-
mutations. Target identification will create a greater ervation of pharmaceutical products. In: Preservatives in the Food.
understanding of microbial survival mechanisms and give Pharmaceutical and Environmental Industries. ed. R.G. Board.
M.C. Allwood and J.G. Banks, pp. 255-279. Blackwell Scientific
leads to their avoidance. Moreover, the association of
Publications, Oxford.
autocidal events with disinfection processes may well Gilbert, P.. Beveridge, E.G.. Crone, P.B., 1977. The lethal action of 2-
offer new avenues for exploration. phenoxyethanol and its analogues upon Escherichia coli NCTC
5933. Microbios. 19. 125-141.
Greenberg, J.T.. Demple. B., 1986. Glutathione in Escherichia coli is
dispensable for resistance to HzOz and gamma radiation. J. Bact.
References 68, 1026-1029.
Hugo, W.B., 1991. A brief history of heat and chemical preservation
Bloomfield, S.F., 1991. Methods for Assessing Antimicrobial Activity. and disinfection. J. Appi. Bacterial. 71, 9918.
In: Mechanisms of Action of Chemical Biocides: Their Study and Hugo, W.B., 1992. Disinfection mechanisms. In: Principles and Practice
Exploitation, ed. S.P. Denyer and W.B. Hugo, pp. l-22. Blackwell of Disinfection, Preservation and Sterihsation. 2nd edition, ed. A.D.
Scientific Publications, Oxford. Russell, W.B. Hugo and G.A.J. Ayliffe, pp. 1877210. Blackwell
Brown, M.R.W.. Collier, P.J.. Gilbert, P., 1990. Influence of growth Scientific Publications, Oxford.
rate susceptibility to antimicrobial agents: modification of the cell Hugo, W.B.. 1995. A brief history of heat, chemical and radiation
envelope and batch and continuous culture studies. Antimicrob. preservation and disinfection. Int. Biodet. Biodeg., pp. 1977217.
Ag. Chemother. 34, 162331628. Hugo. W.B., Denyer, S.P.. 1987. The concentration exponent of dis-
Broxton. P.. Woodcock, P.M.. Gilbert, P., 1984. Interaction of some infectants and preservatives. In: Preservatives in the Food, Phar-
polyhexamethylene biguanides and membrane phospholipids. in maceutical and Environmental Industries, ed. R.G. Board. M.C.
Escherichia coli. J. Appl. Bacterial. 51, 115-124. Allwood and J.G. Banks, pp. 281-292. Blackwell Scientific Pub-
Chapman, J.S.. Diehl. M.A.. 1995. Methylchloroisothiazolone-induced lications, Oxford.
growth inhibition and lethality in Escherichia coli. J. Appl. Bacterial. Hugo. W.B., Russell, A.D., 1992 Types of antimicrobial agents. In:
78, 134141. Principles and Practice of Disinfection. Preservation and Ster-
Daham. S.A.M., Wiseman. D., 1987. The uptake of chlorhexidine by ihsation. 2nd edition., ed. A.D. Russell. W.B. Hugo and G.A.J.
Escherichia co/i and its effects on viability in the absence and pres- Ayliffe, pp. 7788. Blackwell Scientific Publications, Oxford.
ence of EDTA. J. Pharm. Pharmacol.. Suppl. 39. 16P. Kanazawa, A., Ikeda. T.. Endo, T., 1995. A novel approach to mode
Denyer. S.P.. 1990. Mechanisms of action of biocides. Int. Biodet. 26, of action of cationic biocides: morphological effect on antibacterial
899100. activity. J. Appl. Bacterial. 78, 55-60.
Denyer. S.P.. 1995. Mechanisms of action of antibacterial biocides. Int. Karabit, MS., Juneskans, O.T.. Lungren. P.. 1989. Factorial designs
Biodet. Biodeg., pp. 324245. in the evaluation of preservative activity. lnt. J. Pharmaceut. 56,
Denyer. S.P., 1996. Development of preservative systems. In: Microbial 1699174.
Quality Assurance in Cosmetics, Toiletries and Non-sterile Phar- Kroll, R.G.. Anagnostopoulos. G.D., 1981. Potassium leakage as a
maceuticals, ed. R.M. Baird with SF. Bloomfield, pp. 133-148. lethality index of phenol and the effect of solute and water activity.
Taylor and Francis, London. J. Appl. Bacterial. 50, 139-147.
Denyer, S.P.. Hugo, W.B., 1991a. Mechanisms of Action of Chemical Kroll, R.G, Patchett. R.A., 1991. Biocide-induced perturbations of
Biocides: Their Study and Exploitation, ed. S. P. Denyer and W. B. aspects of cell homeostasis: intracellular pH. membrane potential
Hugo. Blackwell Scientific Publications, Oxford. and solute transport. In: Mechanisms of Action of Chemical Bioc-
Denyer, S.P., Hugo, W.B.. 199lb. Biocide-induced damage to the bac- ides: Their Study and Exploitation. ed. S.P. Denyer and W.B. Hugo,
terial cytoplasmic membrane. In: Mechanisms of Action of Chemi- pp. 1899202. Blackwell Scientific Publications, Oxford.
cal Biocides: Their Study and Exploitation. ed. S.P. Denyer and Lambert, P.A., Smith, A.R.W.. 1976. Antimicrobial action of dode-
Hugo. W.B.. pp. 171-188. Blackwell Scientific Publications, Oxford. cyldiethanolamine: activation of ribonuclease I in Escherichia co/i.
Denyer. S.P., Hugo. W.B., Harding, V.D., 1986. The biochemical basis Microbios. 17, 3549.
of synergy between the antibacterial agents, chlorocresol and 2- Leeming. K., Moore, C.P., Denyer, S.P.. 1997. The Bug Beater-a new
phenylethanol. Int. J. Pharmaceut. 29, 29-36. approach to the control of micro-organisms in photoprocessing
Denyer, S.P.. Jackson, D.E., Al-Sagher. M., 1991. Intracellular delivery applications. International Symposium on Silverhalide Imaging.
of biocides. In: Mechanisms of Action of Chemical Biocides: Their Society for Imaging Science and Technology. Victoria, Canada.
Study and Exploitation. ed. S.P. Denyer and W.B. Hugo, pp. 2633 Lehmann, R.H., 1988. Synergisms in disinfectant formulations. In:
270. Blackwell Scientific Publications, Oxford. Industrial Biocides, Critical Reports on Applied Chemistry, ed. K.
DiMonte, D., Bellomo, G.. Thor. H., Nicotera, P., Orrhenius, S., 1984. R. Payne, Vol. 23, pp. 6890. Wiley, Chichester.
Menadione-induced cytoxicity is associated with protein thiol oxi- Martin, S.E.. Flowers, R.S., Ordal, Z.J.. 1976. Catalase: its effect on
dation and alteration in intracellular calcium homeostasis. Arch. microbial enumeration. Appl. Env. Microbial. 32. 7322734.
Biochem. Biophys. 235, 343-350. Nicas. T.I.. Hancock, R.E.W., 1980. Outer membrane protein HI of
Dodd. C.E.R., Sharman. R.L.. Bloomfield. S.F., Booth, I.R., Stewart, P.~eudomonas aeryyinosa: involvement in adaptive and mutational
268 S.P. Denyer and G.S.A.B. Stewartllnternationai Biodeterioration & Biodegradation 41 (1998) 261-268
resistance to ethylenediaminetetraacetate, polymyxin B and gen-
tamicin. J. Bacterial. 143, 872-878.
Paulus, W.. 1991. Microbicides for the protection of materials: yester-
day, today and tomorrow. In: Biodeterioration and Biodegredation,
ed. H.W. Rossmoore, Vol. 8, pp. 35-52. Elsevier Applied Science
Publishers, London.
Paulus, W., 1993 Microbicides for the Protection of Materials: a Hand-
book. Chapman and Hall, London.
Phillips-Jones, M.K., Rhodes-Roberts, M.E., 1991. Studies of inhibi-
tors of respiratory electron transport and oxidative phos-
phorylation. In: Mechanisms of Action of Chemical Biocides: Their
Study and Exploitation. ed. S. P. Denyer and W. B. Hugo, pp. 203-
224. Blackwell Scientific Publications, Oxford.
Reichart, O., 1994. Modelling the destruction of Escherichia coli on the
base of reaction kinetics. Int. J. Food Microbial. 23, 449465.
Richards, R.M.E., Xing, D.K.L.. King. T.P., 1995. Activity of p-amino-
benzoic acid compared with other organic acids against selected
bacteria. J. Appl. Bacterial. 78, 209-215.
Russell, A.D., 1991. Mechanisms of bacterial resistance to non-anti-
biotics: food additives and food and pharmaceutical preservatives.
.I. Appl. Bacterial. 71, 191-201.
Russell, A.D., Morris, A., Allwood, MC.. 1973. Methods for assessing
damage to bacteria induced by chemical and physical agents. Meth.
Microbial. 8, 95-182.
Russell, A.D., Furr, J.R., Maillard, J-Y., 1997. Microbial susceptibility
and resistance to biocides. ASM News. 63,481487.
Salton, M.R.J., Owen, P. 1976. Bacterial membrane structures. Ann.
Rev. Microbial. 30, 451482.
Shepherd, J.A., Waigh, R.D., Gilbert, P., 1988. Antibacterial action of
2-bromo-2-nitropropan-1,3-dial (Bronopol). Antimicrob. Ag.
Chemother. 32, 1693-1698.
Singer, S.J., Nicolson, G.L., 1972. The fluid mosaic model of the struc-
ture of cell membranes. Science. 175, 72&73 1.
Stretton, R.J.. Manson, T.W., 1973. Some aspects of the mode of action
of the antibacterial compound Bronopol (2-bromo-2-nitropropan-
1.3-diol). J. Appl. Bacterial. 36, 61-76.
Tang, C.C., Jackson, H., 1979. Minimal medium recovery of chilled
Salmonella heidelberg. J. Appl. Bacterial. 46, 143-146.
van Netten, P.. van der Zee, H., Mossell, D.A.A., 1984. A note on
catalase enhanced recovery of acid injured cells of gram-negative
bacteria and its consequences for the assessment of the lethality of
L-lactic acid decontamination of raw meat surfaces. J. Appl.
Bacterial. 57, 1699173.
Wright, N.E., Gilbert, P., 1987. Antimicrobial activity of N-alkyl-
trimethylammonium bromides: influence of specific growth rate and
nutrient limitation. J. Pharm. Pharmacol. 39. 685-690.