Download A REVIEW Cationic antiseptics: diversity of action under a common

Journal of Applied Microbiology 2005, 99, 703–715
doi:10.1111/j.1365-2672.2005.02664.x
A REVIEW
Cationic antiseptics: diversity of action under a common
epithet
P. Gilbert and L.E. Moore
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK
2005/0059: received 20 January 2005, revised 14 March 2005 and accepted 15 March 2005
1.
2.
3.
4.
Summary, 703
Perspectives, 703
Background, 704
Cationic antimicrobial agents, 705
4.1 Quaternary ammonium compounds, 706
4.2 Biguanides, 708
1. SUMMARY
Cationic antimicrobials have been in general use within
clinical and domestic settings for over half a century.
Recently, the use of antiseptics and disinfectants has been
questioned in such settings because of the possibility that
chronic exposure of the environment to such agents might
select for less susceptible strains towards these agents and
towards third party antibiotics. Whilst no supportive evidence has emerged from retrospective field studies of high use
environments such debate has tempered new applications for
these molecules. In the clinic, use of antiseptics, together with
products, such as dressings, catheters and sutures, which are
impregnated with biocides has increased. Prominent amongst
these biocides are the cationics. Much of the research
pertaining to the mechanisms of action of cationic antibacterials was conducted in the 1960s and 1970s and has not been
subject to extensive review. Analysis of available publications
suggest that monoquaternary ammonium compounds (QAC,
cetrimide, benzalkonium chloride), biquaternaries and bisbiguanides (Chlorhexidine, Barquat), and polymeric biguanides (Vantocil, Cosmocil) whilst having similarities in action
mechanism, differ substantially in the nature of their
interaction with cell envelopes. This has profound implications in terms of cross-resistance where changes in susceptibility towards QAC is not reflected in changes towards other
cationics. This review examines action mechanisms for these
Correspondence to: Peter Gilbert, School of Pharmacy and Pharmaceutical Sciences,
University of Manchester, Manchester M13 9PL, UK
(e-mail: [email protected]).
ª 2005 The Society for Applied Microbiology
4.2.1 The bisbiguanides chlorhexidine and
alexidine, 709
4.2.2 Polyhexamethylene biguanides, 710
5. Conclusions, 712
6. References, 713
agents and highlights key differences that render them
distinct categories of antibacterial agent.
2. PERSPECTIVES
In recent years there has been some questioning of the
potential of antiseptic residues, accumulating within high use
environments such as clinics and hospitals, to select for
bacteria that are altered with respect to their susceptibility not
only towards other antiseptics but also towards third party
agents such as antibiotics. The utility of cationic antibacterials
to combat cross infection is undeniable, as is the overall
contribution of antisepsis to the reduction of hospitalacquired infection. Indeed, reductions in antibiotic use
brought about by such policies can be argued to have had a
positive impact upon antibiotic resistance development.
Nevertheless, re-examination of the action and resistance
mechanisms associated with all antibacterial molecules destined for use in a clinical and domestic setting is appropriate.
Cationic antimicrobials have been widely deployed in
antisepsis for well over half a century without any apparent
reduction in their effectiveness. They remain the mainstay of
routine chemical antisepsis and disinfection. Amongst the
commonly deployed cationic antimicrobials are the quaternary ammonium compounds (QAC; cetrimide, benzalkonium
chloride), bisbiguanides (chlorhexidine, hibitane) and polymeric biguanides (vantocil). All of these positively charged
molecules bind strongly to the cell walls and membranes of
bacteria because of their opposite, negative charge. Disruption of the target cell is brought about by perturbation of these
704 P . G I L B E R T A N D L . E . M O O R E
sites. The nature of the interaction, following binding,
determines activity and the potential for resistance development. The purpose of this article is to consider the mechanism
of action of cationic antimicrobials, possible resistance
mechanisms and their use in infection control highlighting
key differences that make each group of molecules distinct.
3. BACKGROUND
Problems associated with the development and spread of
antibiotic resistance have been increasing since the early
1960s and, in the clinic, is currently viewed as a major
threat to the treatment of hospital and communityacquired infection (McMurray 1992; Kaatz et al. 1993)
with as many as one-third of nosocomial infections
believed to be preventable (Senior 2001). Such resistance
has generally been associated with the overuse and abuse
of therapeutic agents, and with the acquisition and fusion
of genetic elements encoded within plasmids. It is widely
accepted that the main cause of this problem has been, and
still is widespread inappropriate use and over-prescribing
of antibiotics in clinical medicine, animal husbandry and
veterinary practice (Rao 1998; House of Lords Select
Committee on Science and Technology 1998; Feinman
1999; Georgala 1999; Magee et al. 1999; Dixon 2000).
Concerns about bacterial resistance have led to calls for
increased education, of both public and professionals, on
the correct use of antibiotics. Additionally, more stringent
infection control measures have been advocated in order to
reduce the transmission of infection (Anon 1997, 1999a,b,
1999c; Hart 1998; Cristino 1999; Dwyer 1999; Smith et al.
1999; Waldvogel 1999; Dixon 2000). These measures
recognize the tremendous contributions that antisepsis has
made, over the last century, towards our current advanced
state of public health. Indeed, if reductions in the number
of infections requiring antibiotic treatment can be achieved
through effective hygiene, including the use of antiseptic
products, then this will delay increases in the incidence of
antibiotic resistance. Accordingly, it is important to ensure
that the use of antiseptic products is not discouraged in
situations where it is part of good hygienic practice and
where there may be tangible reductions in the transmission
of infection. With respect to the management of postoperative wounds then, whilst multiple factors related to the
nature of the surgical procedure can influence the risk of
wound infection, their approximate incidence, for clean
procedures, is between 2 and 3% (Futoryan and Grand
1995). The normal flora of the skin is an important source
of serious postoperational infections with the involvement
of skin organisms such as Staphylococcus aureus and
Staphylococcus epidermidis being widely acknowledged.
Furthermore, antibiotic resistant coryneform bacteria have
been isolated from the skin of both hospitalized patients
and control groups (Larson et al. 1986). Systemic infections caused by such bacteria are often associated with
concurrent use of indwelling medical devices such as
central venous lines or catheters (Passerini et al. 1992), but
systemic treatment with vancomycin neither eliminates nor
prevents colonization of the device (Larson et al. 1986).
Topical application of broad-spectrum antimicrobial agents
such as QACs, biguanides, halogen release agents and
triclosan remain safe and effective preventative, and
treatment, measures. In this respect, a chlorhexidine
containing medication was the only formulation to thoroughly eliminate both 1-day and 3-day-old Enterococcus
faecalis biofilms (Lima et al. 2001). Whilst such use has
generally been confined to medicated soaps, handwashes
and bathing formulae, incorporation of these and other
antimicrobial agents within the polymer materials and
coatings that comprise indwelling medical devices and
dressings have demonstrated significant applications in the
localized prevention of infection (Storch et al. 2002).
For over a century cationic antimicrobials have been
used both in infection control and within many consumer
products and have often been assumed to possess a single,
generic mechanism of action directed towards biological
membranes. Cationic antimicrobials that have been in use
for over 40 years include a variety of quaternary ammonium-based molecules (cetrimide, benzalkonium chloride),
bisbiguanides (chlorhexidine) and polymeric biguanides
(VantocilTM; Arch Chemicals, Blackley, UK). Sadly, in
spite of their long and widespread use, assumptions
relating to such agents are compounded by a general lack
of experimental evidence surrounding their biological
mechanisms. Recently the use of such antimicrobial agents
has been questioned in many application areas. This
questioning is based on an association between trace levels
of antimicrobial residue and their implied potential to
select for less susceptible bacteria that are co-incidentally
resistant to third party antibiotics (Gilbert and McBain
2002, 2003). QACs are used extensively in the food
processing industry to prevent the persistence of pathogens
such as Escherichia coli and Listeria monocytogenes in the
environmental microflora (Holah et al. 2002). Biofilm
formation is thought to play an important role in the
survival of virulent strains of food-related staphylococci.
Staphylococci isolated from the food industry were found
to vary greatly in their ability to form biofilms, but biofilm
formation was positively correlated with resistance to
QACs (Moretro et al. 2003). Chlorhexidine is also a
well-known anti-plaque biocide that plays a crucial role
in the reduction of supragingival plaque and treatment of
gingivitis. Large numbers of common oral bacteria isolated
from patients using chlorhexidine indicate no increase in
microbial resistance to chlorhexidine, or to commonly used
antibiotics (Sreenivasan and Gaffar 2002). An effective
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CATIONIC ANTISEPTICS
strategy for the reduction of legionella in water cooling
systems is regular disinfection with polyhexamethylene
biguanide (PHMB) (Kusnetsov et al. 1997).
Although a number of laboratory studies have demonstrated links between the exposure of pure cultures to
sublethal concentrations of biocides and changes in antibiotic and antimicrobial susceptibility (Braoudaki and Hilton
2004; Joynson et al. 2002; Tattawasart et al. 1999), there is
little or no evidence suggesting that this is a significant
factor in the development of antibiotic resistance in clinical
practice (Loughlin et al. 2002; Gilbert and McBain 2003).
The current view is that if biocides do play a role, then it is
likely to be a very minor one but it is a subject that requires
constant review (Russell 2003). Biocides have played a major
part in reducing the number of nosocomial infections
through effective hygiene, so it is important to ensure that
biocide use is not discouraged in situations where there is
real benefit. Conversely, it is also necessary to assess the
possibility that widespread and inappropriate use of biocides
could compromise their in-use effectiveness (Bloomfield
2002; Braoudaki and Hilton 2004).
A number of mechanisms account for the wide range of
sensitivity noted for the antibacterial action of antibiotics
and biocides (Heinzel 1998). Some organisms and genera,
by virtue of the absence of critical targets sites or an
inability of the agents to accumulate at those targets, are
intrinsically resistant to particular groups of agent under all
growth conditions (Hancock 1998). Other groups of organism may undergo phenotypic changes in susceptibility that
reflect the conditions under which they were cultivated or
exposed, the temporary expression of efflux pumps or
synthesis and export of protective enzymes (inductive
change), or mutations in the genes encoding or regulating
a sensitive target site (chromosomal change). Growth as a
biofilm also reduces the susceptibility profile and is probably
caused by a variety of factors including nutrient depletion
within the biofilm, reduced access of the biocide to cells in
the biofilm, chemical interaction between the biocide and
the biofilm, and the production of degradative enzymes and
neutralizing chemicals (Gilbert et al. 1990, 2002; Brown
and Gilbert 1993; McDonnell and Russell 1999; Campanac
et al. 2002).
Polyhexamethylene biguanide is a polymeric cationic
antimicrobial agent that has been deployed in consumer
applications for over 40 years. Recently it has been used in
the treatment of Acanthamoeba keratitis and as an adjunct in
various wound dressing materials. Whilst PHMB shares
many of the attributes of other, simpler, cationic agents, it
has additional actions that render it unique amongst this
generic class of antimicrobials.
The purpose of the current article is to consider the
mechanism of action of the family of cationic antimicrobials,
possible resistance mechanisms and the potential impact of
705
their use on resistance development. The molecular basis of
antimicrobial action will therefore be considered from the
most simple (monoquaternary ammonium compounds),
through bisbiguanides to PHMB. At each stage the potential
for resistance development will be considered against a
background of published susceptibility surveillance articles
(Gilbert and McBain 2003).
4. CATIONIC ANTIMICROBIAL AGENTS
The outermost surface of bacterial cells universally carries a
net negative charge, often stabilized by the presence of
divalent cations such as Mg2+ and Ca2+. This is associated
with the teichoic acid and polysaccharide elements of Grampositive bacteria, the lipopolysaccharide of Gram-negative
bacteria, and the cytoplasmic membrane itself. It is not
therefore surprising that many antimicrobial agents are
cationic and have a high binding affinity for bacterial cells.
Often, cationic antimicrobials require only a strong positive
charge together with a hydrophobic region in order to
interact with the cell surface and integrate into the
cytoplasmic membrane. Such integration into the membrane
is sufficient to perturb growth and at the treatment levels
associated with antiseptic formulations is sufficient to cause
the membrane to lose fluidity and for the cell to die. Indeed,
for many decades such compounds have been loosely
designated as membrane active agents or as biological
detergents broadly recognizing their lack of specificity in
mechanism of action. It is however worthwhile considering
for a moment the general characteristics of microbial
membranes in order to fully understand the mode of action
of cationic antimicrobials. The membranes are composed
primarily of proteins, embedded within a lipid matrix and
approximating to a bilayer (Singer and Nicolson 1972). The
proteins either fully traverse the two sides of the bilayer
(integral proteins) or are peripheral and associate with one
specific side. Many of these membrane proteins are required
in order to maintain the structural integrity of the
membrane, whilst others are functional and associated with
catabolism, cellular transport and the biosynthesis of wall
and extracellular products (toxins, virulence factors, etc.).
The hydrophobic environments of the neighbouring phospholipids moderate the functionalities of these proteins.
Thus, each protein is surrounded by particular phospholipids that interact with the protein. In many cases the
precise nature of the phospholipid head group assists
maintenance of an active configuration for the enzyme.
The lipid bilayer is further stabilized by divalent cations
such as Ca2+. Cationic antimicrobials are relatively hydrophobic but interact initially with the wall and membrane by
displacing these divalent cations. Such action is shared with
simple chelating agents such as EDTA and EGTA that
perturb membrane structure solely through the sequestra-
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706 P . G I L B E R T A N D L . E . M O O R E
tion of stabilizing metal cations. Subsequent interactions of
the cationic biocides with membrane proteins and lipid
bilayer depend upon the specific nature of the biocide. Many
of the cationic antimicrobials have been deployed as surface
and topical antimicrobials, in the clinic and in general hygiene
delivery, for more than half a century. Notable amongst these
agents are the QAC (benzalkonium chloride, cetrimide,
Barquat), bisbiguanides (chlorhexidine) and polymeric
biguanides (VantocilTM, CosmocilTM; Arch Chemicals)
together with antibiotics such as Polymyxin and Tyrocidin.
Of these the quaternary ammonium group of compounds and
the polymeric biguanides are mixtures of compounds that
share a common generic structure. Even within groups such
as the monoquaternaries the use of simple vegetable oils as the
synthetic starting material for their chemical synthesis leads
to the marketed molecules being mixtures with very diverse
compositions with respect to the composition of different
alkyl chain substituents (Daoud et al. 1983; Gilbert and
Al-Taae 1985). Such chemical diversity broaden the
spectrum of activity but makes standardization between
batches and different manufacturers difficult.
In considering the action and utility of the cationic
biocides, together with their potential for resistance development, it is worthwhile characterizing them according to
the number of cationic groupings per molecule. Thus the
QACs are generally monocationic, whilst bisbiguanides
(chlorhexidine) carry two cationic groups separated by a
hydrophobic bridging structure (hexamethylene), and polymeric biguanides are polycationic linear polymers comprising a hydrophobic backbone with multiple cationic
groupings separated by hexamethylene chains.
4.1 Quaternary ammonium compounds
The QACs are amphoteric surfactants, generally containing
one quaternary nitrogen associated with at least one major
hydrophobic substituent (Fig. 1). Cetrimide USP is tetradecyltrimethylammonium bromide whereas the generic term
Cetrimide relates to mixtures of n-alkyltrimethyl ammonium
bromides where the n-alkyl group (the hydrophobic moiety)
is between eight and 18 carbons long. Benzalkonium
chlorides are always mixtures of n-alkyldimethylbenzyl
Fig. 1 Structure of tetradecyldimethylbenzyl ammonium chloride (one of the benzalkonium chlorides where the major n-alkyl group is C14 (a), and
tetradecyltrimethylammonium bromide (Cetrimide USP) (b). Counter-ions are not shown. Positively charged head groups are shaded blue. Major
hydrophobic chains are shaded purple
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CATIONIC ANTISEPTICS
ammonium chlorides where the n-alkyl groups can be of
variable length within a specified range. In addition to these,
various dialkylmethyl ammonium halides and dialkylbenzyl
ammonium halides also have antimicrobial activity and are
variously deployed as biocides, preservatives and antiseptics
(Brannon 1997). All share a common mechanism of action.
The raw materials providing the alkyl group of these
synthetic compounds are often natural oils such as coconut
or soya bean oil. These natural oils are heterogenous
mixtures of fatty acids with carbon chain lengths of between
6 and 22. Whilst 10, 12, 14 and 16 carbon fatty acids
dominate their relative abundance varies from batch to
batch. Commercially available QACs that utilize natural oils
as the source of the alkyl chain substituents will therefore be
highly diversified not only in their fatty-acyl chain length
distributions but also in the degree of C–C saturation. Each
of these factors will significantly affect antimicrobial activity.
The activity of quaternary ammonium biocides is an
approximate parabolic function of the compounds lipophilicity (n-alkyl chain length) (Daoud et al. 1983; Gilbert and
Al-Taae 1985). For Gram-positive bacteria and yeast, such
activity maximizes with chain lengths of n ¼ 12–14, whilst
for Gram-negative bacteria, optimal activity is achieved for
compounds with a chain length of n ¼ 14–16. Compounds
with n-alkyl chain lengths of <n ¼ 4 or >n ¼ 18 are
virtually inactive. As the antimicrobial activity of QACs
towards specific species of bacteria is dependent upon the
hydrophobicity of the n-alkyl chain then, given the raw
material source of the n-alkyl substituent, the overall activity
of individual commercial products can be highly variable
(Daoud et al. 1983; Gilbert and Al-Taae 1985). Many
quaternary ammonium mixtures are however blended so as
to optimize activities against specific groups of bacteria, or to
gain as broad a spectrum of activity as is possible.
Pharmacopoeial standards for such molecules define the
mixture chain lengths, but such standards differ between
regulatory bodies.
The mode of action of QAC against bacterial cells is
thought to involve a general perturbation of lipid bilayer
membranes as found to constitute the bacterial cytoplasmic
membrane and the outer-membrane of Gram-negative
bacteria. Such action leads to a generalized and progressive
leakage of cytoplasmic materials to the environment. Low
concentrations of QAC bind firmly to anionic sites found
on the membrane surface, cause cells both to lose
osmoregulatory capability and to leak potassium ions and
protons (Lambert and Hammond 1973). Intermediate levels
perturb membrane-located physiologies such as respiration,
solute transport, and cell wall biosynthesis (Salt and
Wiseman 1970). The high concentrations used in many
biocidal formulations however, kill cells by solubilization of
the membranes to release all of the cells contents, hence
their designation as biological detergents (Salton 1951,
707
1968). Indeed, the surfactant properties of QACs are often
used to good advantage in disinfectant cleansing formulations (Hugo 1956). At a molecular level, action involves an
association of the positively charged quaternary nitrogen
with the head groups of acidic-phospholipids within the
membrane (Fig. 2b). The hydrophobic tail then interdigitates into the hydrophobic membrane core (Fig. 2b,c).
Thus, at low concentration (approximately minimum
growth inhibitory concentrations, MIC) such interaction
increases the surface pressure in the exposed leaflet of the
membrane to decrease membrane fluidity and phase
transition temperature. The membrane undergoes a transition from fluid to liquid crystalline state and loses many of
its osmoregulatory and physiological functions (Fig. 2d).
The membrane core decreases in hydrophobicity and
phospholipids tend towards a stable hexagonal arrangement. At use-concentrations, solutions of QACs form
mixed micellar aggregates that solubilize hydrophobic
membrane components (i.e. lipid A, phospholipids etc.
see Fig. 2e,f).
The QACs have been actively deployed since the 1930s
with no apparent reduction in their effectiveness. Nevertheless there are numerous reports of apparent resistance
towards QAC. Such resistance invariably refers to changes
in the MIC and do not affect the activity at use-levels
(Gilbert and McBain 2003). The latter are often 100–1000·
higher than the MIC. Where changes in QAC MIC have
been demonstrated then these have either been relatively
minor (2–3 fold) and associated with changes in the acidic
phospholipid content of the membrane (Wright and Gilbert
1987a) or they have been associated with the acquisition, or
hyperexpression of multi-drug efflux pumps (i.e. qac genes)
(Heir et al. 1999). Such efflux pumps can actively remove
QAC from the membrane core and thereby reduce effectiveness at sub-MIC. Acquisition or hyperexpression of
multidrug efflux pumps has however been associated with
changes in MIC of therapeutically important third party
antibiotics which co-incidentally serve as substrates to those
pumps (Poole 2003). Some species of bacteria, notably
Pseudomonas aeruginosa, are relatively insensitive to QAC
biocides. This is thought to relate to a failure of the
compounds to penetrate the outer-membrane and to access
the cytoplasmic membrane. Such insensitivity can often be
overcome by formulating in solutions of EDTA and EGTA.
These sequester divalent cations from the outer and
cytoplasmic membrane and thereby aid interaction with
QAC.
Order of magnitude increases are noted in the antimicrobial activities of n-alkyl-QACs when the n-alkyl chain
length is increased beyond 10. This is related to a
concentration independent dimerization of the molecules
in solution. Above these critical chain lengths attraction
between the adjacent hydrophobic chains exceeds the
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708 P . G I L B E R T A N D L . E . M O O R E
(b)
(a)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
(d)
(c)
Ca++
Ca++
(f)
(e)
Hydrophilic domain
Phospholipids
Protein
Benzalkonium
chloride
Fig. 2 Cartoon showing the mechanism of action for quaternary ammonium biocides. The segments (a–f) show progressive adsorption of the
quaternary headgroup to acidic phospholipids in the membrane with increasing QAC exposure/concentration. This leads to decreased fluidity of the
bilayers and the creation of hydrophilic voids in the membrane. Protein function is perturbed with an eventual lysis of the cell, and solubilization of
phospholipids and proteins into QAC/phospholipid micelles. Inset micrograph shows vesicle formation from outer membrane caused by QAC
treatment
electrostatic repulsion of their charged nitrogen head
groups. QAC dimers are formed that bear bi-polar positive
charges in conjunction with interstitial hydrophobic
regions. Such dimers both interact more strongly with
the cytoplasmic membrane, than the monomeric form of
QAC, and are able to more easily solubilize within it
(Daoud et al. 1983; Gilbert and Al-Taae 1985). Bisbiguanides, such as chlorhexidine, provide similar bi-polar
configurations of cationic and hydrophobic domains within
a single molecule and are potent antibacterial agents.
4.2 Biguanides
Biguanides were first synthesized in the early part of the 20th
century and have since provided a variety of drugs with a
broad range of pharmacological activities (anti-malarial,
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blood-sugar lowering, antiseptic, anti-protozoal). Marked
antibacterial activity was noted for mixtures of PHMB salts
produced by the reaction of hexamethylene bis-dicyanodiamide and hexamethylene diamine (Rose and Swain 1956).
PHMB (Fig. 3) produced by this route contains polydisperse
oligomers with molecular weights of between 500 and 6000,
in which n varies from 2 to 20. The mixtures have a weight
average of n of 5Æ5 with the tetramer being the dominant
species. Due to the method of synthesis each member of the
series might have either amine or cyanoguanidine groups at
either end position. Attempts to rationalize the PHMB
mixtures were unsuccessful at that time, and precluded their
use in pharmaceutical products. Further synthesis led to the
development of the closely related bisbiguanides. In this
series of molecules, optimal antibacterial activity was exhib-
709
ited by the bisbiguanide 1,6-bis(4¢-chlorophenylbiguanide)
hexane (Davies et al. 1954) (Fig. 4a). This molecule
became marketed as chlorhexidine. Alexidine (Fig. 4b), a
related molecule with ethylhexyl end-groups replacing the
4-chlorophenol endgroups was developed for its activity
against plaque-forming organisms (Spolsky and Forsythe
1977).
4.2.1 The bisbiguanides chlorhexidine and alexidine.
Chlorhexidine, is active towards a wide range of Grampositive and Gram-negative bacteria and is compatible with
a variety of commonly used antibiotics. Whilst the molecule
had little systemic activity in mice, it was found to be highly
effective against wounds infected with haemolytic streptococci (Rose and Swain 1956).
Fig. 3 Generalized structure for polyhexamethylene biguanide (PHMB) chloride counter-ion not shown. N average chain length is 5Æ5 with the
tetramer dominating in the mixture. The end groups are randomly dispersed
Fig. 4 Chemical structures of the bisbiguanides chlorhexidine (a) and alexidine (b). Cationic phospholipid binding sites are indicated by blue
shading. Hydrophobic hexamethylene group indicated by purple shading
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710 P . G I L B E R T A N D L . E . M O O R E
Chlorhexidine has since been widely deployed in surgical
handwashes, as an antiseptic and in various topical treatments for wound sepsis. Chlorhexidine has also been
marketed extensively within various oral hygiene products
as an anti-plaque agent, and within topical slow release
vehicles for the treatment of periodontal disease (Hope and
Wilson 2004). There have been few if any reports of
chlorhexidine resistance at use concentrations, in spite of its
widespread use for almost 50 years in clinical and domestic
settings, but small changes (c. fivefold) in MIC have been
noted (Thomas and Stickler 1979; Kropinski et al. 1982).
The latter is thought to relate to changes in envelope
composition particularly with regard to anionic targets and
cation binding rather than to target modification and or
efflux (Kropinski et al. 1982; Wright and Gilbert 1987b).
Notable, in the spectrum of activity of bisbiguanides is
however their ineffectiveness against some Gram-negative
bacteria particularly Pseudomonadaceae and Providentia spp.
(Thomas and Stickler 1979). As with the QAC biocides such
insensitivity can often be overcome by formulating together
with a chelating agent such as EDTA.
Bisbiguanides antiseptics have a very similar mechanism
of action to the QAC biocides in that the biguanide
groupings associate strongly with exposed anionic sites on
the cell membrane and cell wall, particularly acidic phospholipids and proteins (Chawner and Gilbert 1989b,c).
Binding to such sites is stronger than that of the QAC’s
and can causes displacement of wall and membrane
associated divalent cations (Mg2+, Ca2+) (Davies 1973;
Jensen 1975). A major difference between the bisbiguanides
and QAC biocides is that the hydrophobic regions of the
QAC biocides become solubilized within the hydrophobic
core of the cell membrane whilst those of chlorhexidine do
not. Being six carbons long, rather than 12–16 carbons, the
hydrophobic region of chlorhexidine is somewhat inflexible
and incapable of folding sufficiently to interdigitate into the
bilayer. Chlorhexidine therefore bridges between pairs of
adjacent phospholipid headgroups each being bound to a
biguanide moiety and displaces the associated divalent
cations (Davies 1973; Fig. 5).
Interestingly the distance between phospholipid headgroups in a closely packed monolayer is roughly equivalent to the length of a hexamethylene grouping. A
bisbiguanide would therefore be capable of binding to two
adjacent phospholipid headgroups. Such binding is critical
for the bisbiguanides as activity is reduced significantly if
the polymethylene bridge is made longer or shorter than
six carbons (Davies et al. 1954). An interaction with the
cell membrane, such as this, will reduce membrane
fluidity at low concentrations and affect the osmoregulatory and metabolic capability of the cell membrane and its
contained enzymes (Hugo and Longworth 1966). These
effects have been variously reported as cellular leakage of
potassium ions and protons (Hugo and Longworth 1964,
1965, 1966; Rye and Wiseman 1968; Elferink and Booij
1974), and inhibition of respiration and solute transport.
At higher, in-use, concentrations, the interactions are
more severe and cause the membrane to adopt a liquid
crystalline state, lose its structural integrity and allow
catastrophic leakage of cellular materials (Longworth 1971;
Chawner and Gilbert 1989a,b, 1989c). Whilst the action of
multi-drug efflux pumps is able to moderate the action of
QAC’s at low concentrations they have no effect upon
the action of bisbiguanides. This is presumably because
the bisbiguanides do not become solubilized within the
membrane core.
4.2.2 Polyhexamethylene biguanides. As antibacterials,
PHMB (Fig. 3) was recognized as possessing superior
antimicrobial effect to other cationic biocides, but it could
only be poorly defined chemically. Early attempts to
rationalize the PHMB mixtures were unsuccessful and
precluded their use in pharmaceutical products. Nevertheless, PHMB was marketed as a broad-spectrum antimicrobial
agent in a number of diverse applications. These included
their use as swimming pool sanitizers (BaquacilTM; Arch
Chemicals) and as preservatives of plasticized PVC (VanquishTM; Arch Chemicals), as well as well as general-purpose
environmental biocides and antiseptics. The antimicrobial
activity of PHMB is superior to that of the bisbiguanide
molecules subsequently derived from it. Analysis of the
antimicrobial activity of PHMB reveals an enigma because,
when mixtures were purified with respect to polymer chain
length, it was observed that antimicrobial activity increased
dramatically, on a mass basis (i.e. lg ml)1), with increasing
polymer chain length (Broxton et al. 1983). Thus, the amineended dimers, corresponded crudely to the bisbiguanides,
but were only poorly active, whilst high molecular weight
materials with n > 10 were highly effective. Clearly whilst
there were broad similarities between the actions of chlorhexidine and PHMB (Davies et al. 1968; Davies and Field
1969) the latter had additional properties that rendered it a
superior biocide. This included a general lack of toxicology
rendering the molecule suitable for use in diverse applications such as swimming pools, beer glass sanitizer together
with a lack of colour, taste and surfactancy.
A schematic representation of the interactions of PHMB
with the cell membrane is given in Fig. 6. As with the
bisbiguanides, PHMB was shown to bind rapidly to the
envelope of both Gram-positive and Gram-negative bacteria
and in doing so displaces the otherwise stabilizing presence
of Ca2+ (Broxton et al. 1984a). This binding is to the
cytoplasmic membrane itself, and also to lipopolysaccharide
and peptidoglycan components of the cells wall. The
hexamethylene bridging groups of the polymer, as with
the bisbiguanides, are hydrophobic yet sufficiently inflexible
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CATIONIC ANTISEPTICS
(a)
Ca++
(b)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
(d)
(c)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Chlorhexidine
Phospholipids
711
Hydrophilic domain
Protein
Fig. 5 Diagramatic representation of the interaction of chlorhexidine with the bacterial cytomplasmic membrane. Diagram shows progressive
decreases in fluidity of the outer leaflet with increasing exposure to the bisbiguanide
that they cannot interdigitate into the hydrophobic core of
the cell membrane. Once again, therefore, a bridging of
adjacent acidic phospholipids is brought about by interaction of the antimicrobial with the cell membrane (Broxton
et al. 1984c; Ikeda et al. 1985a,b). One additional feature of
this interaction is that it will tend to become concentrated
around any points of maximum charge density within the
membrane (Ikeda et al. 1984a,b). It has been shown that
integral proteins constitute such sites. Thus, the initial
interactions of PHMB and the membrane will be concentrated around such proteins, leading to a loss of their
function by inflicting changes in their boundary phospholipid environment. This manifests itself, as with the
bisbiguanides, as a loss of transport, biosynthetic and
catabolic capability. The unique polymeric nature of PHMB
means that, unlike for the bisbiguanide, such bridging is not
restricted to pairs of adjacent phospholipids. Rather adsorption to the cell membrane will lead to a sequestration of
common acidic phospholipids into domains comprised of
single rather than mixtures of the phospholipids (Broxton
et al. 1984a,b, 1984c; Ikeda et al. 1985a,b). Thus, in the
presence of PHMB the homogeneous distribution of
phospholipids normally associated with biological membranes is transformed into a mosaic of individual phospholipid domains. Each of these will have different phase
transition properties causing the membrane to fragment into
fluid and liquid crystalline regions. As with other cationic
biocides this is manifested as a generalized cellular leakage,
first of small cationic materials such as potassium ions and
later by losses of intracellular pool materials (Broxton et al.
1983). A secondary consequence of domain formation is that
the separated phospholipid types will assume their energet-
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712 P . G I L B E R T A N D L . E . M O O R E
(a)
(b)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
(d)
(c)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
PHMB
Phospholipids
Hydrophilic domain
Protein
Fig. 6 Diagrammatic representation of the mechanism of action of PHMB directed against a bacterial cytoplasmic membrane. The diagram
illustrates a progressive interaction of PHMB with the acidic membrane components leading to a loss of fluidity and eventual phase separation of the
individual lipids. Individual domains then undergo a transition to the more stable hexagonal arrangement, leading to membrane dissolution
ically favoured position of a hexagonal phase leading to a
total loss of the membrane permeability barrier (Ikeda et al.
1985b). The ability of PHMB to create single lipid domains
within heterogeneous lipid-bilayers is clearly a function of
polymer chain length with longer polymers being able to
form the larger domains and hence the greater perturbation
of membrane function (Ikeda 1991).
The PHMB therefore not only embodies the attributes of
the bisbiguanides in terms of antimicrobial action but
possesses additional molecular activity. As with chlorhexidine there is no evidence that PHMB susceptibility is
affected by the induction or hyperexpression of multi-drug
efflux pumps, neither have there been any reports of
acquired resistance towards these agents. Rather, as with all
membrane active antimicrobials, small changes in MIC have
been reported that correlate with alterations in envelope
lipid composition and cation binding (Broxton et al. 1984c;
Das et al. 1998).
5. CONCLUSIONS
The PHMB was the forerunner of the highly successful
antiseptic agent chlorhexidine, and falls into a general
category of antibacterial agents that are cationic, displace
divalent cations from the wall and membrane of bacteria and
bring about a disruption of the lipid bilayer. The biguanides
and polymeric biguanides differ from other cationic biocides
in that they interact only superficially with the lipid bilayer
altering fluidity through cation displacement and headgroup bridging. QACs on the contrary interact fully with the
membrane and are therefore susceptible to resistance
mechanisms mediated through multidrug efflux pumps.
The activity of biguanides and bisbiguanides is unaffected
by such hyperexpression of efflux. Their deployment in the
clinic, or indeed within consumer products, cannot therefore
have implications towards the selection of multi-resistant
organisms through this mechanism (Gilbert and McBain
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CATIONIC ANTISEPTICS
2003). The multiplicity of critical lethal targets affected by
the interaction of biguanides with the membrane dictate
against singular mutations leading to changes in susceptibility (Gilbert and McBain 2002, 2003). This is borne out by
a lack of evidence to suggest that the use of either compound
over 40 years has affected their activity in the field.
The toxicity profile with regard to skin irritancy and
hypersensitivity of both the biguanides and the polymeric
biguanides is excellent at typical in-use levels. Whilst the use
of PHMB has until recently been restricted to use as a
nonspecific disinfectant (Swimming pool sanitizer, beer glass
sanitizer, antimicrobial fabric conditioner), such restriction
relates to difficulties in standardization of PHMB formulations rather than to toxicity issues. Second generation
PHMB formulations such as Cosmocil CQTM give a much
better definition of polymer dispersity than was previously
possible with PHMB. Such PHMB formulations are now
widely deployed in clinical applications such as the treatment of Acanthamoeba keratinitis (Larkin et al. 1992) and
have been included within certain contact lens cleansing
formulations. PHMB has also been used as an antiseptic for
various applications in medicine and wound care (Larkin
et al. 1992; Wagner 1995; Willenegger et al. 1995; Kramer
and Behrens-Baumann 1997).
With respect to the deployment of PHMB as part of a
wound care system there is little or no evidence to suggest
that this would lead to the emergence of PHMB resistant
nor antibiotic resistant strains of nosocomial pathogen. Use
of the agent within a barrier wound dressing such as Kerlix
AMD would impair the growth and penetration through the
dressing of adventitious pathogens both from the environment to the dressed wound and from the wound to healthcare workers and other human contacts. Such action can
only contribute to breaking the cycle of nosocomial infection
and will inevitably reduce the usage of antibiotics currently
used in the care of such infections.
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