Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology
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possesses two aldehyde groupings which are highly reactive and their presence is an
important component of biocidal activity. The monomeric molecule is in equilibrium
with polymeric forms, and the physical conditions of temperature and pH have a
significant effect on this equilibrium. At a pH of 8, biocidal activity is greatest but
stability is poor due to polymerization. By contrast, acid solutions are stable but
considerably less active, although as temperature is increased, there is a breakdown in
the polymeric forms which exist in acid solutions and a concomitant increase in free
active dialdehyde, resulting in better activity. In practice, glutaraldehyde is generally
supplied as an acidic 2% aqueous solution, which is stable on prolonged storage. This
is then 'activated' prior to use by addition of a suitable alkylating agent to bring the pH
of the solution to its optimum for activity. The activated solution will have a limited
shelf-life, in the order of 2 weeks, although more stable formulations are available.
Glutaraldehyde is employed mainly for the cold, liquid chemical sterilization of medical
and surgical materials that cannot be sterilized by other methods. Endoscopes, including
for example, arthroscopes, laparoscopes, cystoscopes and bronchoscopes may be
decontaminated by glutaraldehyde treatment. Contact times employed in practice for
high level disinfection are often considerably less than the many hours recommended
by manufacturers to achieve sterilization. The British Association of Urological Surgeons
recommends that cystoscopes be routinely immersed for at least 10 minutes but that
this should be increased to 1 hour if mycobacterial infection is known or suspected.
Similarly, the British Thoracic Society recommends immersion of bronchoscopes for
20 minutes between immunocompetent patients one hour with immunocompromised
patients to avoid opportunistic mycobacteria. Whilst M. tuberculosis is successfully
eliminated from instruments after 1 hour with 2% glutaraldehyde, M. avium-intracellulare
strains take much longer to inactivate as they are as much as 12 times more resistant to
glutaraldehyde than M. tuberculosis. Gastroscopes from HIV-positive patients are
required by the British Society of Gastroenterology to be immersed in 2% glutaraldehyde
for 1 hour.
3.3.2 Formaldehyde
Formaldehyde (HCHO) can be used in either the liquid or gaseous state for disinfection
purposes. In the vapour phase it has been used for decontamination of safety cabinets
and rooms; however, recent trends have been to combine formaldehyde vapour with
low temperature steam (LTSF) for the sterilization of heat-sensitive items (Chapter
20). Formaldehyde vapour is highly toxic and potentially carcinogenic if inhaled,
thus its use must be carefully controlled. It is not very active at temperatures below
20°C and requires a relative humidity of at least 70%. The agent is not supplied as a
gas but as either a solid polymer, paraformaldehyde, or a liquid, formalin, which is a
34-38% aqueous solution. The gas is liberated by heating or mixing the solid or
liquid with potassium permanganate and water. Formalin, diluted 1:10 to give 4%
formaldehyde, may be used for disinfecting surfaces. In general, however, solutions of
either aqueous or alcoholic formaldehyde are too irritant for routine application to
skin, while poor penetration and a tendency to polymerize on surfaces limit its use as a
disinfectant.
Chemical disinfectants, antiseptics and preservatives 215
3.3.3
Formaldehyde-releasing agents
Various formaldehyde condensates have been developed to reduce the irritancy
associated with formaldehyde while maintaining activity and these are described as
formaldehyde-releasing agents or masked-formaldehyde compounds.
Of these, noxythiolin (N-hydroxy-Af-methylthiourea) has the greatest pharmaceutical
use as an antimicrobial agent. The compound is supplied as a dry powder and on aqueous
reconstitution slowly releases formaldehyde and iV-mefhylthiourea. Antimicrobial
activity is considered to be due to both the noxythiolin molecule and the released
formaldehyde. Noxythiolin is used both topically and in accessible body cavities as an
irrigation solution and in the treatment of peritonitis. The compound has extensive
antibacterial and antifungal properties.
Polynoxylin (poly[methylenedi(hydroxymethyl)urea]) is a similar compound
available in gel and lozenge formulations.
Taurolidine (bis-[l,l-dioxoperhydro-l,2,4-thiadiazinyl-4] methane) is a condensate
of two molecules of the amino acid taurine and three molecules of formaldehyde. It is
more stable than noxythiolin in solution and has similar uses. The activity of taurolidine
is stated to be greater than that of formaldehyde.
3.4 Biguanides
3.4.1 Chlorhexidine and alexidine
Chlorhexidine is an antimicrobial agent first synthesized at Imperial Chemical
Industries in 1954 in a research program to produce compounds related to the biguanide
antimalarial, proguanil. Compounds containing the biguanide structure could be
expected to have good antibacterial effect; thus, the major part of the proguanil structure
is found in chlorhexidine. The chlorhexidine molecule, a bisbiguanide, is symmetric. A
hexamethylene chain links two biguanide groups to each of which a p-chlorophenyl
radical is bound (Fig. 10.3). A related compound is the bisbiguanide alexidine which
has use as an oral antiseptic and antiplaque agent. Alexidine (Fig. 10.3A) differs from
chlorhexidine (Fig. 10.3B) in that it possesses ethylhexyl end-groups.
Fig. 10.3 Bisbiguanides: A, alexidine; B, chlorhexidine.
216 Chapter 10
Chlorhexidine base is not readily soluble in water therefore the freely soluble salts,
acetate, gluconate and hydrochloride, are used in formulation. Chlorhexidine exhibits
the greatest antibacterial activity at pH 7-8 where it exists exclusively as a di-cation.
The cationic nature of the compound results in activity being reduced by anionic
compounds including soap and many anions due to the formation of insoluble salts.
Anions to be wary of include bicarbonate, borate, carbonate, chloride, citrate and
phosphate with due attention being paid to the presence of hard water. Deionized or
distilled water should preferably be used for dilution purposes. Reduction in activity
will also occur in the presence of blood, pus and other organic matter.
Chlorhexidine has widespread use, in particular as an antiseptic. It has significant
antibacterial activity though Gram-negative bacteria are less sensitive than Gram-
positive. A concentration of 1:2000000 prevents growth of, for example, Staph, aureus
whereas a 1:50000 dilution prevents growth of Ps. aeruginosa. Reports of pseudomonad
contamination of aqueous chlorhexidine solutions have prompted the inclusion of small
amounts of ethanol or isopropanol. Chlorhexidine is ineffective at ambient temperatures
against bacterial spores and M. tuberculosis. A limited antifungal activity has been
demonstrated which unfortunately restricts its use as a general preservative. Skin
sensitivity has occasionally been reported, although, in general, chlorhexidine is well
tolerated and non-toxic when applied to skin or mucous membranes and is an important
preoperative antiseptic.
3.4.2 Polyhexamethylene biguanides
The antimicrobial activity of chlorhexidine, a bisbiguanide, exceeds that of monomeric
biguanides. This has stimulated the development of polymeric biguanides containing
repeating biguanide groups linked by hexamethylene chains. One such compound is a
commercially available heterodisperse mixture of polyhexamethylene biguanides
(PHMB, polyhexanide) having the general formula shown in Fig. 10.4, where n varies
with a mean value of 5.5. The compound has a broad spectrum of activity against
Gram-positive and Gram-negative bacteria and has low toxicity. PHMB is employed
as an antimicrobial agent in various ophthalmic products.
3.5 Halogens
Chlorine and iodine have been used extensively since their introduction as disinfecting
agents in the early 19th century. Preparations containing these halogens such as Dakin's
solution and tincture of iodine were early inclusions in many pharmacopoeiae and
national formularies. More recent formulations of these elemens have improved activity,
stability and ease of use.
Chemical disinfectants, antiseptics and preservatives 217
3.5.1 Chlorine
A large number of antimicrobially active chlorine compounds are commercially
available, one of the most important being liquid chlorine. This is supplied as an amber
liquid by compressing and cooling gaseous chlorine. The terms liquid and gaseous
chlorine refer to elemental chlorine whereas the word 'chlorine' is normally used to
signify a mixture of OCl~, Cl
2
, HOC1 and other active chlorine compounds in aqueous
solution. The potency of chlorine disinfectants is usually expressed in terms of parts
per million (ppm) or percentage of available chlorine (avCl).
3.5.2 Hypochlorites
Hypochlorites are the oldest and remain the most useful of the chlorine disinfectants
being readily available and inexpensive. They exhibit a rapid kill against a wide spectrum
of microorganisms including fungi and viruses. High levels of available chlorine will
enable eradication of acid-fast bacilli and bacterial spores. The compounds are
compatible with most anionic and cationic surface-active agents and are relatively
inexpensive to use. To their disadvantage they are corrosive, suffer inactivation by
organic matter and can become unstable. Hypochlorites are available as powders or
liquids, most frequently as the sodium or potassium salts of hypochlorous acid (HOC1).
Sodium hypochlorite exists in solution as follows:
NaOCl + H
2
0 ^ HOC1 + NaOH
Undissociated hypochlorous acid is a strong oxidizing agent and its potent
antimicrobial activity is dependent on pH as shown:
HOCl^H
+
+ OCl-
At low pH the existence of HOC1 is favoured over OC1" (hypochlorite ion). The
relative microbiocidal effectiveness of these forms is of the order of 100:1. By lowering
the pH of hypochlorite solutions the antimicrobial activity increases to an optimum at
about pH 5; however, this is concurrent with a decrease in stability of the solutions.
This problem may be alleviated by addition of NaOH (see above equation) in order to
maintain a high pH during storage for stability. The absence of buffer allows the pH to
be lowered sufficiently for activity on dilution to use-strength. It is preferable to prepare
use-dilutions of hypochlorite on a daily basis.
3.5.3 Organic chlorine compounds
A number of organic chlorine, or chloramine, compounds are now available for
disinfection and antisepsis. These are the N-chloro (=N-C1) derivatives of, for example,
sulphonamides giving compounds such as chloramine-T and dichloramine-T and
halazone (Fig. 10.5), which may be used for the disinfection of contaminated drinking
water.
A second group of compounds, formed by N-chloro derivatization of heterocyclic
compounds containing a nitrogen in the ring, includes the sodium and potassium salts
of dichloroisocyanuric acid (e.g. NaDCC). These are available in granule or tablet
218 Chapter 10
form and, in contrast to hypochlorite, are very stable on storage, if protected from
moisture. In water they will give a known chlorine concentration. The antimicrobial
activity of the compounds is similar to that of the hypochlorites when acidic conditions
of use are maintained. It is, however, important to note that where inadequate ventilation
exists, care must be taken not to apply the compound to acidic fluids or large spills of
urine in view of the toxic effects of chlorine production. The Health and Safety Executive
(HSE) has set the occupational exposure standard (OES) short-term exposure limit at
1 ppm (see section 2.5 also).
3.5.4 Chloroform
Chloroform (CHC1
3
) has a narrow spectrum of activity. It has been used extensively as
a preservative in pharmaceuticals since the last century though recently has had
limitations placed on its use. Marked reductions in concentration may occur through
volatilization from products resulting in the possibility of microbial growth.
3.5.5 Iodine
Iodine has a wide spectrum of antimicrobial activity. Gram-negative and Gram-positive
organisms, bacterial spores (on extended exposure), mycobacteria, fungi and viruses
are all susceptible. The active agent is the elemental iodine molecule, I
2
. As elemental
iodine is only slightly soluble in water, iodide ions are required for aqueous solutions
such as aqueous iodine solution, BP 1988 (Lugol's Solution) containing 5% iodine in
10% potassium iodide solution. Iodine (2.5%) may also be dissolved in ethanol (90%)
and potassium iodide (2.5%) solution to give weak iodine solution, BP 1988 (Iodine
Tincture).
The antimicrobial activity of iodine is less dependent than chlorine on temperature
and pH, though alkaline pH should be avoided. Iodine is also less susceptible to
inactivation by organic matter. Disadvantages in the use of iodine in skin antisepsis are
staining of skin and fabrics coupled with possible sensitizing of skin and mucous
membranes.
3.5.6 Iodophors
In the 1950s iodophors (iodo meaning iodine and phor meaning carrier) were developed
to eliminate the side-effects of iodine while retaining its antimicrobial activity. These
allowed slow release of iodine on demand from the complex formed. Essentially, four
generic compounds may be used as the carrier molecule or complexing agent. These
give polyoxymer iodophors (i.e. with propylene or ethyene oxide polymers), cationic
(quaternary ammonium) surfactant iodophors, non-ionic (ethoxylated) surfactant
iodophors and polyvinylpyrrolidone iodophors (PVP-I or povidone-iodine). The
Chemical disinfectants, antiseptics and preservatives 219
non-ionic or cationic surface-active agents act as solubilizers and carriers, combining
detergency with antimicrobial activity. The former type of surfactant especially, produces
a stable, efficient formulation the activity of which is further enhanced by the addition
of phosphoric or citric acid to give a pH below 5 on use-dilution. The iodine is present
in the form of micellar aggregates which disperse on dilution, especially below the
critical micelle concentration (cmc) of the surfactant, to liberate free iodine.
When iodine and povidone are combined, a chemical reaction takes place forming
a complex between the two entities. Some of the iodine becomes organically linked to
povidone though the major portion of the complexed iodine is in the form of tri-iodide.
Dilution of this iodophor results in a weakening of the iodine linkage to the carrier
polymer with concomitant increases in elemental iodine in solution and antimicrobial
activity.
The amount of free iodine the solution can generate is termed the 'available iodine'.
This acts as a reservoir for active iodine releasing it when required and therefore largely
avoiding the harmful side-effects of high iodine concentration. Consequently, when
used for antisepsis, iodophors should be allowed to remain on the skin for 2 minutes to
obtain full advantage of the sustained-release iodine.
Cadexomer-I
2
is an iodophor similar to povidone-iodine. It is a 2-hydroxymethylene
crosslinked (1-4) a-D-glucan carboxymethyl ether containing iodine. The compound
is used especially for its absorbent and antiseptic properties in the management of leg
ulcers and pressure sores where it is applied in the form of microbeads containing
0.9% iodine.
3.6 Heavy metals
Mercury and silver have long been known to have antibacterial properties and
preparations of these metals were among the earliest used antiseptics, but have been
replaced by less toxic compounds. Other metals such as zinc, copper, aluminium and
tin have weak antibacterial properties but are used in medicine for other functions, e.g.
aluminium acetate and zinc sulphate are employed as astringents.
3.6.1 Mercurials
The organomercurial derivatives which are still in use in pharmacy are thiomersal and
phenlymercuric nitrate or acetate (PMN or PMA) (Fig. 10.6).
Thiomersal is employed as a preservative for eye-drops and in lower concentration,
0.001-0.004%, as a preservative for contact lens solutions. The phenylmercuric salts
(0.002%) are also used for preservation of eye-drops but long-term use has led to
220 Chapter 10
keratopathy and they are not recommended for prolonged use. Use of both mercurials
has declined considerably due to risk of hypersensitivity and local irritation. They are
absorbed from solution by rubber closures and plastic containers to a significant extent.
Hydrogen peroxide and peroxygen compounds
The germicidal properties of hydrogen peroxide (H
2
0
2
) have been known for more
than a century, but use of low concentrations of unstable solutions did little for its
reputation. However, stabilized solutions are now available and due to its unusual
properties and antimicrobial activity, hydrogen peroxide has a valuable role for specific
applications. It is used as an antiseptic for open wounds and ulcers where it provides
additional cleansing due to its oxidation of organic debris. Its activity against the
protozoa, Acanthamoeba, which can cause keratitis in contact lens wearers, has made
it popular for disinfection of soft contact lenses. Concentrations of 3-6% are effective
for general disinfection purposes. At high concentrations (up to 30%) and increased
temperature hydrogen peroxide is sporicidal. Use has been made of this in vapour-
phase hydrogen peroxide decontamination of laboratory equipment and enclosed spaces.
Peracetic acid (CH
3
COOOH) is the peroxide of acetic acid and is a more potent
biocide than hydrogen peroxide, with excellent rapid biocidal activity against bacteria,
including mycobacteria, fungi, viruses and spores. It can be used in both the liquid and
vapour phases and is active in the presence of organic matter. It is finding increasing
use at concentrations of 0.2-0.35% as a chemosterilant of medical equipment. Its
disadvantages are that it is corrosive to some metals. It is also highly irritant and must
be used in an enclosed system.
Of the other peroxygen compounds with antimicrobial activity, potassium
monoperoxysulphate is the main product marketed for disinfectant use. It is used for
body fluid spillages and equipment contaminated with body fluids, but its activity against
mycobacteria and some viruses is limited.
Phenols
Phenols (Fig. 10.7) are widely used as disinfectants and preservatives. The phenolics
for disinfectant use have good antimicrobial activity and are rapidly bactericidal but
generally are not sporicidal. Their activity is markedly diminished by dilution and is
also reduced by organic matter. They are more active at acid pH. The main disadvantages
of phenols are their caustic effect on skin and tissues and their systemic toxicity. The
more highly substituted phenols are less toxic and can be used as preservatives and
antiseptics; however, they are also less active than the simple phenolics, especially
against Gram-negative organisms.
Phenol (carbolic acid)
Phenol no longer plays any significant role as an antibacterial agent. It is of historical
interest, since it was introduced by Lister in 1867 as an antiseptic and has been used as
a standard for comparison with other disinfectants, which are then given a phenol
coefficient in tests such as the Rideal-Walker test.
Chemical disinfectants, antiseptics and preservatives 221
Fig. 10.7 Structural formulae of phenolic disinfectants: A, clear soluble fluids; B, black and white
fluids; C, chlorinated phenols; D, bisphenols.
3.8.2
Tar acids
Many of the phenols which are used in household and other commercial disinfectant
products are produced from the tar obtained by distillation of coal or more recently
petroleum. They are known as the tar acids. These phenols are separated by fractional
distillation according to their boiling point range into phenol, cresols, xylenols and
high boiling point tar acids. As the boiling point increases the properties of the products
alter as shown:
Phenols
Cresols
Xylenols
High boiling point
Tar acids
Boiling point increases
Bactericidal activity increases
Inactivation by organic matter increases
Water solubility decreases
Tissue toxicity decreases
The phenols from the higher boiling point fractions have greater antimicrobial activity
but must be formulated so as to overcome their poor solubility. A range of solubilized
and emulsified phenolic disinfectants are available including the clear soluble fluids,
black fluids and white fluids.
Clear soluble fluids. Cresol is a mixture of o-, m- and p-methyl phenol (Fig.
10.7A). Because of its poor solubility, it is solubilized with a soap prepared from
linseed oil and potassium hydroxide. It forms a clear solution on dilution. This
preparation, known as Lysol (Cresol and Soap Solution BP 1968) has been widely
used as a general purpose disinfectant but has largely been superseded by less irritant
phenolics.
By using a higher boiling point fraction than cresols, consisting of xylenols and
ethylphenols (Fig. 10.7A), a more active, less corrosive product which retains activity
in the presence of organic matter, is obtained. It is also solubilized with a soap to
give a clear soluble fluid. A variety of proprietary products for general disinfection
purposes are available with these phenols as active ingredients. They possess rapid
bactericidal activity, including mycobacteria, providing a major use for the terminal
disinfection of rooms occupied by patients with open tuberculosis. Similarly, further
use is made of these compounds for spills of faeces containing pathogens such as
salmonellae and shigellae and for controlling outbreaks of methicillin-resistant Staph.
aureus (MRSA).
Black fluids and white fluids. Black fluids and white fluids are prepared by solubilizing
the high boiling point tar acids (Fig. 10.7B). Black fluids are homogenous solutions,
which form an emulsion on dilution with water. White fluids are finely dispersed
emulsions of tar acids, which on dilution with water produce more stable emulsions
than do black fluids. Both types of fluid have good bactericidal activity. Preparations
are very irritant and corrosive to the skin and are strong smelling; however, they are
relatively inexpensive and are useful for household and general disinfection purposes.
They must be used in adequate concentrations as activity is reduced by organic matter
and is markedly affected by dilution.
Chemical disinfectants, antiseptics and preservatives 223
3.8.3 Non-coal tar phenols (chloroxylenol and chlorocresol)
Many derivatives of phenol are now made by a synthetic process. Homologous series
of substituted derivatives have been prepared and tested for antimicrobial activity. A
combination of alkyl substitution and halogenation has produced useful derivatives
including clorinated phenols which are constituents of a number of proprietary
disinfectants. Two of the most widely used derivatives are/?-chloro-m-cresol (4-chloro-
3-methylphenol, chlorocresol, Fig. 10.7C) which is mostly employed as a preservative
at a concentration of 0.1%, and /?-chloro-m-xylenol (4-chloro-3,5-dimethylphenol,
chloroxylenol, Fig. 10.7C) which is used for skin disinfection, although less than
formerly. Chloroxylenol is sparingly soluble in water and must be solubihzed, for
example in a suitable soap solution in conjunction with terpineol or pine oil. Its
antimicrobial capacity is weak and is reduced by the presence of organic matter.
3.8.4 Bisphenols
Bisphenols are composed of two phenolic groups connected by various linkages.
Hydroxy halogenated derivatives, such as hexachlorophane (Fig. 10.7D) and triclosan,
are the most active microbiologically, but are bacteriostatic at use-concentrations and
have little antipseudomonal activity. The use of hexachlorophane is also limited by its
serious toxicity. Both hexachlorophane and trichlosan have limited application in
medicated soaps and washing creams.
3.9 Surface-active agents
Surface-active agents or surfactants are classified as anionic, cationic, non-ionic or
ampholytic according to the ionization of the hydrophilic group in the molecule. A
hydrophobic, water-repellent group is also present. Within the various classes a range
of detergent and disinfectant activity is found. The anionic and non-ionic surface-active
agents, for example, have strong detergent properties but exhibit little or no antimicrobial
activity. They can, however, render certain bacterial species more sensitive to some
antimicrobial agents, possibly by altering the permeability of the outer envelope.
Ampholytic or amphoteric agents can ionize to give anionic, cationic and zwiterionic
(positively and negatively charged ions in the same molecule) activity. Consequently,
they display both the detergent properties of the anionic surface-active agents and the
antimicrobial activity of the cationic agents. They are used quite extensively in Europe
for pre-surgical hand scrubbing, medical instrument disinfection and floor disinfection
in hospitals.
Of the four classes of surface-active agents, however, the cationic compounds
arguably play the most important role in an antimicrobial context.
3.9.1 Cationic surface-active agents
The cationic agents used for their antimicrobial activity all fall within the group known
as the quaternary ammonium compounds which are variously described as QACs, quats
or onium ions. These are organically substituted ammonium compounds as shown in
224 Chapter 10