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A REVIEW

Cationic antiseptics: diversity of action under a commonepithet

P. Gilbert and L.E. MooreSchool 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. 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 evi-

dence 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 antibac-

terials 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 bis-

biguanides (Chlorhexidine, Barquat), and polymeric bigua-

nides (Vantocil, Cosmocil) whilst having similarities in action

mechanism, differ substantially in the nature of their

interaction with cell envelopes. This has profound implica-

tions in terms of cross-resistance where changes in suscep-

tibility towards QAC is not reflected in changes towards other

cationics. This review examines action mechanisms for these

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 hospital-

acquired 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 des-

tined 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 quater-

nary ammonium compounds (QAC; cetrimide, benzalkonium

chloride), bisbiguanides (chlorhexidine, hibitane) and poly-

meric biguanides (vantocil). All of these positively charged

molecules bind strongly to the cell walls and membranes of

bacteria because of their opposite, negative charge. Disrup-

tion of the target cell is brought about by perturbation of these

1. Summary, 703

2. Perspectives, 703

3. Background, 704

4. Cationic antimicrobial agents, 705

4.1 Quaternary ammonium compounds, 706

4.2 Biguanides, 708

4.2.1 The bisbiguanides chlorhexidine and

alexidine, 709

4.2.2 Polyhexamethylene biguanides, 710

5. Conclusions, 712

6. References, 713

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

Journal of Applied Microbiology 2005, 99, 703–715 doi:10.1111/j.1365-2672.2005.02664.x

sites. The nature of the interaction, following binding,

determines activity and the potential for resistance develop-

ment. The purpose of this article is to consider themechanism

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 community-

acquired 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 postop-

erative 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 infec-

tions 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), butsystemic 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 thor-

oughly eliminate both 1-day and 3-day-old Enterococcusfaecalis 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 ammo-

nium-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

704 P. GILBERT AND L.E. MOORE

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 703–715, doi:10.1111/j.1365-2672.2005.02664.x

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 demon-

strated links between the exposure of pure cultures to

sublethal concentrations of biocides and changes in antibi-

otic 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 organ-

ism 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

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 Gram-

positive 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 �biologicaldetergents� 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 phosp-

holipids moderate the functionalities of these proteins.

Thus, each protein is surrounded by particular phosphol-

ipids 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 hydro-

phobic 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-

CATIONIC ANTISEPTICS 705


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 devel-

opment, 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 poly-

meric biguanides are polycationic linear polymers compri-

sing 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 tetra-

decyltrimethylammonium 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



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 lipophi-

licity (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 activityof 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,

1968). Indeed, the surfactant properties of QACs are often

used to good advantage in disinfectant cleansing formula-

tions (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 interdigi-

tates 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 trans-

ition 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 arrange-

ment. 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. Never-

theless 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 effect-

iveness 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 antimi-

crobial activities of n-alkyl-QACs when the n-alkyl chainlength 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



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). Bisbigu-

anides, 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,

Ca++

Ca++ Ca++

Ca++ Ca++Ca++

Ca++Ca++ Ca++ Ca++

Ca++

Benzalkoniumchloride

Hydrophilic domain

Phospholipids Protein

(e)

(c)

(a) (b)

(d)

(f)

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



blood-sugar lowering, antiseptic, anti-protozoal). Marked

antibacterial activity was noted for mixtures of PHMB salts

produced by the reaction of hexamethylene bis-dicyanodia-mide 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-

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 Gram-

positive 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 strepto-

cocci (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



Chlorhexidine has since been widely deployed in surgical

handwashes, as an antiseptic and in various topical treat-

ments 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 phosp-

holipids 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 head-

groups in a closely packed monolayer is roughly equiv-

alent 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 osmoregula-

tory 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. Neverthe-

less, 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 (Van-

quishTM; 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 amine-

ended 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 chlorh-

exidine 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 applica-

tions 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



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 interac-

tion of the antimicrobial with the cell membrane (Broxton

et al. 1984c; Ikeda et al. 1985a,b). One additional feature ofthis 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 concen-

trated around such proteins, leading to a loss of their

function by inflicting changes in their boundary phospho-

lipid 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 adsorp-

tion 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 mem-

branes is transformed into a mosaic of individual phosphol-

ipid 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-

Phospholipids Protein

Chlorhexidine Hydrophilic domain

Ca++Ca++ Ca++

Ca++

Ca++Ca++

Ca++

Ca++ Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++Ca++

Ca++Ca++

Ca++

Ca++Ca++

Ca++

(c) (d)

(a) (b)

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



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 chlorhexi-

dine 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 head-

group 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

Phospholipids

Hydrophilic domain PHMB

Protein

Ca++Ca++ Ca++

Ca++

Ca++Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++

Ca++Ca++

Ca++

Ca++

Ca++Ca++

(c) (d)

(a) (b)

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



2003). The multiplicity of critical lethal targets affected by

the interaction of biguanides with the membrane dictate

against singular mutations leading to changes in suscepti-

bility (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 formula-

tions 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 treat-

ment 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 environ-

ment to the dressed wound and from the wound to health-

care 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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